ES2908046T3 - Anti-siglec-15 antibodies and uses thereof. - Google Patents

Abstract

An isolated antibody or antigen-binding fragment thereof that specifically binds Siglec-15 comprising the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 sequences of Table 1 corresponding to SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

Description

DESCRIPTION

Anti-Siglec-15 antibodies and uses thereof

BACKGROUND

Disclosure field

The present disclosure relates to anti-Siglec-15 immunoconjugates, antibodies, antigen-binding fragments, and uses thereof, particularly in the treatment of acute myeloid leukemia.

Disclosure Background

Acute myeloid leukemia (AML) is a fatal disease in which normal hematopoiesis is replaced by a malignant proliferation of immature blast cells. Symptoms such as infection, bleeding, and anemia are related to this bone marrow failure and, if left untreated, lead to the death of the patient. AML affects approximately 2,000 people a year in the UK, with the incidence increasing as the population ages. Curative treatment is very intensive, which is arduous for the patient, expensive and requires prolonged hospital stays, which represents a significant cost for the national health system. In addition, the use of allogeneic bone marrow transplantation, either initially or as salvage therapy, represents an additional cost in those patients in whom it is appropriate. Despite this, the majority of AML patients continue to die from the disease.

Many drugs have been developed to treat AML. In addition to conventional chemotherapeutic agents, therapeutic antibodies have been developed for selective targeting of AML blasts. Gemtuzumab ozogamicin (GO), which targets a Siglec sialic acid-binding immunoglobulin-like protein, CD33, is approved for patients older than 60 years (Bross PF et al., Clin. Cancer Res. 7(6):1490- 6 (2001)).

CD33 is an antigen found on many AML blasts, and GO is a toxin-conjugated antibody that binds to CD33. Recently, a randomized trial (AML15) reported a disease-free survival advantage for patients treated with GO, mainly related to a lower incidence of relapse (Burnett AK et al., J. Clin. Oncol. 29(4):369-77 (2011)). However, approximately 10% of AML cases do not express CD33, and GO is also associated with relatively rare but life-threatening side effects, including myelosuppression, perhaps because CD33 is expressed on healthy hematopoietic stem cells ( Taussig DC et al., Blood 106(13): 4086-92 (2005)). In the AML15 trial, this side effect appears to have been manifested by the fact that patients treated with OG required more platelet transfusions and intravenous antibiotics. In other studies, GO treatment has also resulted in hepatotoxicity as a dose-limiting side effect (Mulford D., Semin. Hematol. 45(2):104-9 (2008)).

Although the existence of cancer stem cells in all malignant neoplasms is a hotly debated topic, it is widely accepted that they are present in leukemia. The first proof of the existence of cancer stem cells was demonstrated in acute myeloid leukemia. Bonnet and Dick isolated the CD34+CD38- subpopulation of leukemic cells and showed that these cells could repopulate tumors in NOD/SCID mice (Bhatia M. et al., Proc. Natl. Acad. Sci. 94(10):5320-5 (1997)). Recent work also suggests that leukemia stem cell potential is present in the CD38+ compartment (Taussig DC et al., Blood 112(3): 568-75 (2008)). With a growing understanding of their biology and unique identity, work is beginning to turn to leukemic stem cells (Chan WI and Huntly BJ, Semin. Oncol. 35(4):326-35 (2008)). In leukemia therapy, active research is targeting this subpopulation of leukemic stem cells, however, there are no drugs available to date that specifically target these cells.

Siglecs are a family of sialic acid-binding immunoglobulin superfamily receptors (Varki A and Angata T, Glycobiology 16(1):1R-27R (2006)). They are commonly expressed on immune cells, particularly immune cells of the myeloid lineage. Most Siglecs are inhibitory, but novel activating Siglecs have recently been discovered, namely Siglec-14, 15 and 16 (Angata T et al., FASEB J. 20:1964-1973 (2006), Angata T et al., Glycobiology 17:838-846 (2007), Cao H et al., Eur J Immunol 38:2303-2315 (2008)). Different Siglecs have varying affinities for bonds other than sialic acids, which are nine-carbon sugars found on the periphery of most mammalian cell surfaces. Siglec-15 is a recently described Siglec that is well conserved between species with recognizable orthologs of the human sequence in zebrafish. Initial characterization of Siglec-15 revealed that its ligand is 2,6-linked sialic acids, the binding of which depended on an essential arginine residue in the N-terminal V-set immunoglobulin domain of Siglec-15 (Angata T et al ., 2007). Siglec-15 has also been shown to bind to an antigen highly expressed on a variety of tumors, sialyl-Tn (Angata T et al., 2007). Siglec-15 is unusual in that it is endowed with both negative and positive signaling motifs. In the transmembrane domain, Siglec-15 can associate with positive signaling adapter molecules, such as DAP10, DAP12, and the Fc receptor common y-chain, but, at the same time, Siglec-15 encodes an ITIM-like cytoplasmic motif known as immunoreceptor tyrosine-based switch motif (ITSM) that generally mediates inhibitory signals (Shlapatska LM et al., J Immunol. 166(9):5480-7 (2001)). The only Another example of an immune receptor encoding dual signaling motifs is KIR2DL4, which associates with ITAM encoding the Fc receptor common adapter chain (Miah SM et al., J. Immunol., 180(5):2922-32 (2008)); Kikuchi-Maki A et al., J. Immunol., 174(7):3859-63 (2005)) and also contains an inhibitory ITIM motif in its own cytoplasmic tail (Faure M and Long EO, J. Immunol., 168 (12):6208-14 (2002)).

The interest in Siglec as targets for the treatment of leukemia is increasing. Nguyen et al. profiled most of the known CD33rSiglecs (a major subfamily of Siglecs) for expression on AML cells. (Nguyen DH, Exp. Hematol., 34(6):728-35 (2006)) They identified Siglec expression profiles characteristic of each of the major subgroups of AML, as well as patient-specific CD33rSiglec fingerprints. A personalized approach to leukemia treatment has been proposed in which a complete profile of a patient's Siglec expression is performed and followed by targeting to a combination of Siglec. The advantage of Siglec targeting is that most of them exhibit rapid endocytosis. Antibody-induced endocytosis of Siglec-5 and Siglec-9 on the surface of U937 cells have similar half-lives of approximately 100 minutes. However, Siglec-based therapies have not been developed for the treatment of AML. Angata T et al., (2007) identifies and characterizes the Siglec-15 protein. O'Reilly M K et al., Trends Pharmaco. Sci. 30(5):240-248 (2009) identifies Siglecs as attractive targets for cell-directed therapies. Hiruma Y et al., Biochem. Biophys. Res. Commun. 409(3):424-429 (2011) identifies Siglec-15 as a regulator of osteoclast differentiation. US 2002/0110862 identifies Siglec-like gene (SLG) and SLG-related proteins, and discloses methods of treating cancer and hematopoietic disorders by modulating the biological activity of a related protein. with the SLG.

BRIEF SUMMARY

An association between Siglec-15 expression and leukemia is described herein. The invention provides immunoconjugates, antibodies and antigen-binding fragments thereof against Siglec-15 and methods of manufacturing said antibody or antigen-binding fragments thereof, and the use of said molecules to treat AML according to claims . The invention also provides isolated cells, polynucleotides, vectors and compositions according to the claims. Importantly, immunoconjugates, antibodies, and antigen-binding fragments against Siglec-15 provide a therapeutic treatment that avoids the side effects associated with CD33-targeted anti-AML therapies.

Disclosed herein is an isolated antibody or antigen-binding fragment thereof that specifically binds to Siglec-15 comprising: a VH sequence that is at least 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 1; a VL sequence at least 90%, 95% or 100% identical to the amino acid sequence of SEQ ID NO: 2; an identical or identical VH-CDR1, VH-CDR2 and/or VH CDR3 sequence except for one, two or three substitutions in each CDR relative to the VH-CDR1, VH-CDR2 and VH-CDR3 sequences corresponding to SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively; an identical or identical VL-CDR1, VL-CDR2 and/or VL CDR3 sequence except for one, two or three substitutions in each CDR with respect to the VL-CDR1, VL-CDR2 and VL-CDR3 sequences corresponding to SEQ ID NO: 6, SEQ ID NO: 7 and Se Q ID NO: 8, respectively; or identical or identical VH-CDR1, VH-CDR2, VH CDR3, VL-CDR1, VL-CDR2, and VL CDR3 sequences except for one, two, or three substitutions in each CDR relative to the VH-CDR1, VH- CDR2, VH CDR3, VL-CDR1, VL-CDR2 and VL CDR3 corresponding to SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

In particular disclosures, the isolated antibody or antigen-binding fragment thereof comprises a VH sequence at least 90%, 95% or 100% identical to the amino acid sequence of SEQ ID NO: 1 and a VL sequence at least 90%, 95% or 100% identical to the amino acid sequence of SEQ ID NO: 2.

In certain disclosures, an immunoconjugate, isolated antibody, or antigen-binding fragment of the invention induces Siglec-15 endocytosis at a rate where the half-life of endogenous surface Siglec-15 on K562 human myelogenous leukemia cells is less than about 5 minutes, less than about 4 minutes, or less than about 3 minutes

In certain disclosures, an isolated antibody or antigen-binding fragment of the invention specifically binds to, or competes with, the same epitope of Siglec-15 as an antibody or antigen-binding fragment as described above. In further disclosures, the isolated antibody or antigen-binding fragment of the invention specifically binds to the same epitope of Siglec-15 as an antibody or antigen-binding fragment thereof that comprises the VH and/or VL regions, or one or more CDR regions, of A9E8 or competitively inhibits binding to Siglec-15 by an antibody or antigen-binding fragment thereof comprising the VH and/or VL regions, or one or more CDR regions, of A9E8.

In certain disclosures, an antibody or antigen-binding fragment of the invention is humanized, chimeric, fully human, a Fab fragment, a Fab' fragment, an F(ab)2 fragment, or a single-chain Fv (scFv) fragment.

Also disclosed is a polypeptide comprising the VH and/or VL sequences or one or more of the CDR sequences of an antibody or antigen-binding fragment of the invention. An isolated cell that produces the antibody is disclosed, antigen-binding fragment or polypeptide of the invention and a method of manufacturing the antibody, antigen-binding fragment or polypeptide of the invention, comprising (a) culturing an isolated cell that produces the antibody or antigen-binding fragment; and (b) isolating an antibody, antigen-binding fragment, or polypeptide from the cultured cell.

Also disclosed is an immunoconjugate having the formula (A)-(L)-(C) or (C)-(L)-(A), wherein: (A) is an antibody or antigen-binding fragment thereof , or polypeptide of the invention; (L) is a linker; and (C) is a cytotoxic agent; and wherein the linker (L) joins (A) to (C).

Disclosed herein is a composition comprising an immunoconjugate, antibody, antigen-binding fragment, or polypeptide of the invention and a carrier.

Also disclosed is an isolated polynucleotide comprising a nucleic acid encoding a VH or VL as described above. In particular disclosures, the polynucleotide of the invention encodes an antibody comprising the nucleotide sequence SEQ ID NO: 3 or SEQ ID NO: 4. A vector comprising a polynucleotide of the invention is disclosed.

Also disclosed is a method of treating acute myeloid leukemia in a subject in need thereof, comprising administering to the subject an effective amount of an immunoconjugate, antibody, antigen-binding fragment, or composition of the invention. In additional disclosures, a method of treating acute myeloid leukemia reduces the number of leukemic stem cells.

Also disclosed is a method of reducing side effects associated with the treatment of acute myeloid leukemia in a subject in need thereof, comprising administering to the subject an effective amount of an immunoconjugate, antibody, antigen-binding fragment, or composition of the invention. . In certain disclosures, the side effects are myelosuppression and/or hepatotoxicity.

Also disclosed is a method of reducing the number of blasts in a patient with AML comprising administering to the patient an effective amount of an immunoconjugate, antibody, antigen-binding fragment, or composition of the invention. In other disclosures, the blasts are CD33+ or CD33-.

Also disclosed is a method of treating acute myeloid leukemia in a subject who has low expression of CD33 or who is CD33-, comprising administering to the subject an effective amount of an immunoconjugate, antibody, antigen-binding fragment, or antigen composition. the invention. In certain disclosures, the low expression is due to treatment with a CD33-targeted therapy.

Also disclosed is a method of making acute myeloid leukemia therapies comprising generating antibodies that bind to Siglec-15, where the therapies preferentially target AML blasts rather than normal peripheral blood leukocytes. In particular disclosures, the antibodies according to this method are recombinant.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURE

Figure 1: Siglec-15 transcript expression. (A) Four splice variants of human Siglec-15 were identified by RT-PCR using specific primers. Electrophoresis of RT-PCR products from DAMI (megakaryocytic cell line), HEL (erythroleukemic cell line), and K562 (erythroleukemic cell line) are shown (left). Exon maps of the transcripts were generated after sequencing (right). The labeling reflects the number of extracellular immunoglobulin-like domains contained in each splice variant, eg "2D" means 2 domains, etc. For the splice variant of a "1D" domain, two isoforms were identified. "1D-A" encodes an additional short peptide from exon 2, which is missing in "1D-B". (B) Results of RT-PCR analysis identifying a Siglec-15 transcript in 6 of 7 different cell lines tested. Only YT (natural killer cell-like leukemic cell line) did not show any Siglec-15 transcripts. Stimulation with PMA or LPS for 48 hours upregulated the expression of the transcripts. GAPDH RT-PCR is shown below each panel to control for equal charge (0.4 kb). 293T cells were used as a negative control.

Figure 2: Siglec-15 specific monoclonal antibodies. (A) Phage display technology was used to select clones expressing single chain variable fragment (ScFv) binders for purified Siglec-15 and also for cell surface transfected Siglec-15. The variable domains (Vh and VL) of two selected phage ScFvs were cloned into a mouse IgG1 antibody backbone and expressed as whole mouse IgG1 antibodies. The resulting monoclonal antibodies showed stronger binding to the stable cell line CHO-pDisplay Siglec-15 (open histograms) than to the parent CHO cell line (filled histograms) by flow cytometry. HA staining was used as a positive control. The pDisplay constructs encode only the extracellular domains of Siglec-15 with the N-terminal HA tag and a PDGFR transmembrane domain. (B) HEK-293 DAP10 stable cell lines were transiently transfected with full-length FLAG-Siglec-15 for detection by confocal microscopy using monoclonal antibodies A9E8 and A4C9 (red color). The surface marker cell, CD44 (rat antibody, blue color) and the endosomal marker, EEA1 (rabbit antibody, green color) were used to define cell boundaries. Both antibodies stained similarly compared to the positive control (mouse anti-FLAG monoclonal antibody). A stable cell line HEK-293 expressing DAP10 was used because the membrane adapter molecule DAP10 facilitated surface expression of Siglec-15 (FIG. 3). (C) Specificity of A9E8 and A4C9 for Siglec-15 by FMAT (similar to confocal microscopy) comparing different Siglec-encoding stable CHO cell lines. The ScFv versions of A9E8 and A4C9 specifically bound the stable cell line Siglec-15 CHO. (D) Western blot using the A4C9 antibody. The lanes of the gel contain lysates from untransfected 293T cells (UT) and 293T cells transfected (T) with full-length FLAG-Siglec-15 (right). The major band detected at ~43 kDa matches the size of fully glycosylated Siglec-15 and is sensitive to Endo-H digestion. Faint background bands were observed at -50 kDa.

Figure 3: Intracellular expression of Siglec-15. Daudi and K562 cells were subjected to FACS analysis for ^{Siglec-15 expression using monoclonal antibody A9E8.} FACs ^{plots show} forward scatter (FSC) on the x-axis and isotype or Siglec-15 (A9E8) staining on the y-axis. Percentages indicate the proportion of the live cell population with positive antibody staining levels above background (rectangular analysis windows). Unfixed staining shows surface expression (left), while fixed and permeabilized staining (right) reveals additional intracellular expression. For intracellular staining, cells were first fixed in paraformaldehyde (30 minutes, room temperature), washed three times in PBS, and then permeabilized in 0.5% Triton-100 (10 minutes, room temperature) and dried. washed three additional times in PBS. Mouse IgG1 monoclonal isotype control antibody (Dako) was used in parallel. For unfixed cells, live cell gating was performed using SyTox Red stain (Invitrogen).

Figure 4: Adapter association of Siglec-15. FACS analysis of the expression of full-length FLAG-tagged Siglec-15 and 16 on the surface of HEK-293 cells stably expressing different membrane adapter molecules, each expressing HA N-markers, was evaluated. terminals. HA and Flag markers were stained together. The x-axis shows surface adapter expression levels (HA-FITC staining) and the y-axis shows surface Siglec expression (Flag-cy3). The percentage of double positive cells (shown in the upper right quadrant) reflects adapter-dependent surface expression of Siglec. Mock transfection was performed using an empty FLAG-CMV vector. Non-transfected HEK-293 cells were also used as a negative control. Adapter and Siglec pairings that demonstrated increased surface Siglec detection are boxed.

Figure 5: Surface expression of endogenous Siglec-15 in leukemic cell lines. (A) FACS plots of surface Siglec-15 protein expression in leukemic cell lines. The x-axis shows forward scatter (FSC) and the y-axis isotype-matched control or Siglec-15 staining. A9E8 was used for Siglec-15 staining and mouse IgG1 monoclonal control antibody (Dako) as isotype control. Live cell gating was performed using SyTox Red stain (Invitrogen). Percentages indicate the proportion of the live cell population that stain positive at levels above background. Leukemic cell lines of the myeloid lineage are shown in the first two rows. First row: monocytic (U937 and SKM-1) and promonocytic (NOMO-1) cell lines. Second row: erythroleukemic (K562 and HEL) and megakaryocytic (dAMi) cell lines. ^{Third row: B-cell lymphoma (Daudi and Raji) and T-cell acute lymphoblastic leukemia (Jurkat) cell lines} . (B) Western blot analysis of Siglec-15 cytoplasmic tail-specific rabbit antisera (left) and mouse anti-FLAG monoclonal antibody staining of untransfected 293T cell lysates and construct-transfected 293T cell lysates. FLAG-Siglec-15 full length. A fully glycosylated Siglec-15 band was discovered at 43 kDa. (C) Western blot analysis of lysates from four leukemic cell lines (K562, Raji, Jurkat, and HEL) using both the monoclonal antibody A4C9 that recognizes the extracellular domains of Siglec-15 and the Tail 1A antiserum that is specific for the portion of Siglec-15. cytoplasmic tail. Arrows indicate bands from both blots that match the expected size of ~43 kDa for fully glycosylated Siglec-15.

Figure 6: Siglec-15 is expressed in only a small number of a subset of healthy peripheral blood leukocytes. (A) Peripheral blood leukocytes obtained from healthy donors were used to analyze Siglec-15 cell surface expression in different types of leukocyte cells. Murine IgG1 monoclonal antibody (Dako) was used as isotype control and different cell markers were used to differentiate cell lineages: CD3 (T cells), CD19 (B cells), CD56 (NK cells), and CD14 (monocytes). Live cells were gated by removing cells with low forward and side scatter values. The percentages indicate the proportions of live cells in each quadrant. The X-axis corresponds to FITC-labeled lineage marker staining and the y-axis corresponds to the isotype-matched control of Siglec-15 staining. No lineage stained for surface Siglec-15 except <4% of CD14+ (CD14) monocytes. (B) Macrophages and dendritic cells were differentiated from peripheral blood monocytes and used for detection of cell surface Siglec-15. Markers (CD14 for macrophages and CD86) were used to ensure differentiation success. Histograms show unstained (transparent) and marker-stained (red) macrophages and dendritic cells. FACS plots show forward scatter in the x-axis and isotype staining or Siglec-15 (A9E8) on the y-axis. Percentages indicate the percentage of the population with positive staining above background levels. Treated macrophages and dendritic cells are labeled with or without lPs for 24 hours.

Figure 7: Elevated surface expression of Siglec-15 in blasts from AML patients. Blasts from AML patients were used to study the surface expression of Siglec-15. Eight of the 10 patients showed significant Siglec-15 surface expression compared to healthy PBLs. Four examples are shown: (A) FACS analysis of cells from an individual AML patient (Patient A) showing high levels of Siglec-15 expression (35.7% of the population), co-expressing with CD14 and HLA-DR . Mouse IgG1 isotype control was added for comparison. (B) FACS analysis of cells from a second AML patient (Patient B), showing that cells were negative for CD33 while displaying elevated expression of Siglec-15 in 20.2% of cell populations. Histograms compare CD33 expression (unstained - red, stained - clear) and Siglec-15 expression (unstained - red, stained - clear) in the same sample. (C) FACS analysis of cells from a third patient (Patient C) showed 10% of blasts expressing Siglec-15, the majority of which were CD33+CD14+HLA-DR-. (D) FACS analysis of cells from a fourth patient (Patient D) had 14% blasts positive for Siglec-15. Histograms show the difference in Siglec-15 surface staining for larger (higher forward scatter) and smaller (lower forward scatter) blasts (Isotype - red color; Siglec-15 -clear). (E) Summary of the percentage of total circulating cells expressing Siglec-15 from AML patients (dots) or from healthy donors (crosses). Horizontal lines indicate mean values.

Figure 8: Surface endocytosis of Siglec-15. The rate of endocytosis for endogenously expressed surface Siglec-15 on K562 cells was measured by following the loss of surface A9E8 binding after incubation at 37°C. A9E8 staining was performed on ice for 30 minutes. Cells were then resuspended in serum-free medium prewarmed at 37°C and incubated for varying lengths of time at 37°C. Time points ranged from 30 seconds to 15 minutes. An isotype control antibody (mIgG1 control, Dako) was used to measure background staining/fluorescence. Mean fluorescence above isotype staining was calculated for different time points and expressed as a percentage of maximum surface staining without incubation at 37°C. The graph (top) shows the drop in Siglec-15 surface staining over 15 minutes; the x-axis is the time measured in seconds and the y-axis is the percentage of initial fluorescence. An exponential decay curve fits the data points well, corresponding to a half-life of 174 seconds. An experiment was performed in parallel using CHO cells stably expressing surface Siglec-15 in a pDisplay construct that lacks the natural Siglec-15 transmembrane domains and cytoplasmic tail and instead encodes a Siglec-15 transmembrane domain. PDGFR. Consistent levels of surface staining (shown by the line of best fit) for the stable cell line CHO-Siglec-15 demonstrate that A9E8 did not significantly dissociate from the cell surface during the first 15 min of incubation at 37 °C. .

DETAILED DESCRIPTION

I. DEFINITIONS

It should be noted that the expression "a" or "an" entity refers to one or more of that entity; for example, "an antibody that specifically binds Siglec-15" is understood to mean one or more antibodies that specifically bind Siglec-15. Therefore, the terms "a" (or "an"), "one or more" and "at least one" may be used interchangeably herein.

"Siglec-15" is a type I transmembrane protein comprising two immunoglobulin-like domains, a transmembrane domain containing a lysine residue and a short cytoplasmic tail. The extracellular domain of Siglec-15 preferentially recognizes Neu5Aca2-6GalNAca structures and has immune system activating activity.

As used herein, the term "polypeptide" is intended to encompass a "polypeptide" in the singular, as well as "polypeptides" in the plural, and refers to a molecule composed of monomers (amino acids) linked linearly by amide bonds (also known as polypeptides). known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Therefore, peptides, dipeptides, tripeptides, oligopeptides, "protein", "chain of amino acids" or any other term used to refer to a chain or chains of two or more amino acids, are included in the definition of "polypeptide", and the term "polypeptide" may be used in place of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by amino acids. of non-natural origin. A polypeptide may be derived from a natural biological source or may be produced by recombinant technology, but is not necessarily translated from an indicated nucleic acid sequence. It can be generated in any way, including by chemical synthesis.

A polypeptide as disclosed herein can be about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more in size. , 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such a structure. Polypeptides with a defined three-dimensional structure are called folded, and those that do not have a defined three-dimensional structure, but can take on a large number of different conformations, are called unfolded. As used herein, the term "glycoprotein" refers to a protein coupled to at least one carbohydrate moiety that is attached to the protein through an oxygen-containing or nitrogen-containing side chain of an amino acid residue. , for example, a serine residue or an asparagine residue.

By an "isolated" polypeptide or a fragment, variant or derivative thereof is meant a polypeptide that is not found in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are native or recombinant polypeptides that have been partially or substantially separated, fractionated, or purified by any suitable technique.

Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the above polypeptides, and any combinations thereof. The terms "fragment", "variant", "derivative" and "analog" when referring to an antibody that specifically binds Siglec-15, as disclosed herein, include any polypeptide that retains at least some of the antigen-binding properties of the corresponding native antibody or polypeptide. Polypeptide fragments include, for example, proteolytic fragments, as well as suppression fragments, in addition to the specific antibody fragments discussed elsewhere herein. Variants of an antibody that specifically binds Siglec-15 as disclosed herein include fragments as described above and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions or insertions. The variants may be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be produced using mutagenesis techniques known in the art. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of an antibody or antigen-binding fragment that specifically binds Siglec-15, as disclosed herein, are polypeptides that have been altered to exhibit additional characteristics not found in the native polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogs." As used herein, a "derivative" of an antibody or antigen-binding fragment that specifically binds Siglec-15 refers to a subject polypeptide that has one or more residues chemically derivatized by reaction of a functional side group. . Also included as "derivatives" are those peptides that contain one or more naturally occurring amino acid derivatives of the twenty conventional amino acids. For example, 4-hydroxyproline can be substituted for proline; 5-hydroxylysine can substitute for lysine; 3-methylhistidine can be substituted for histidine; homoserine can substitute for serine; and ornithine can substitute for lysine.

The term "polynucleotide" is intended to encompass a single nucleic acid, as well as multiple nucleic acids, and refers to an isolated nucleic acid molecule or construct, eg, messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide may comprise a conventional phosphodiester bond or an unconventional bond (eg, an amide bond, such as found in peptide nucleic acids (PNAs)). The term "nucleic acid" refers to any one or more nucleic acid segments, eg, DNA or RNA fragments, present in a polynucleotide. By "isolated" nucleic acid or polynucleotide is meant a nucleic acid molecule, DNA or RNA, that has been removed from its native environment. For example, a recombinant polynucleotide encoding an antibody or antigen-binding fragment that specifically binds to Siglec-15 contained in a vector is considered isolated as disclosed herein. Additional examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or polynucleotides purified (partially or substantially) in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides. Isolated polynucleotides or nucleic acids further include such synthetically produced molecules. In addition, a polynucleotide or nucleic acid may be or may include a regulatory element, such as a promoter, ribosome binding site, or transcription terminator.

As used herein, a "coding region" is a portion of nucleic acid consisting of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) does not translate into an amino acid, it can be considered part of a coding region, but any flanking sequences, e.g., promoters, ribosome binding sites, transcriptional terminators , introns and the like are not part of a coding region. Two or more coding regions may be present in a single polynucleotide construct, eg, in a single vector, or in separate polynucleotide constructs, eg, in separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, eg, a single vector may separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or nucleic acid may encode heterologous coding regions, either fused or unfused to a nucleic acid encoding an antibody, or antigen-binding fragment, variant, or derivative thereof that specifically binds Siglec-15. . Heterologous coding regions include, without limitation, specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

In certain disclosures, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a polypeptide-encoding nucleic acid may typically include a promoter and/or other transcriptional or translational control elements operatively associated with one or more coding regions. An operative association is when a coding region for a gene product, eg, a polypeptide, is associated with one or more regulatory sequences such that expression of the gene product is under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in transcription of mRNA encoding the desired gene product and if the nature of the junction between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct expression of the gene product nor does it interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operatively associated with a nucleic acid encoding a polypeptide if the promoter were capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcriptional control elements, in addition to a promoter, eg, enhancers, operators, repressors, and transcription termination signals, may be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein.

A variety of transcriptional control regions are known to those of skill in the art. These include, without limitation, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments of cytomegalovirus (immediate early promoter, together with intron A), simian virus 40 (the early promoter) and retroviruses (such as Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit p-globin, as well as other sequences capable of controlling gene expression in cells. eukaryotes. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as promoters inducible by lymphokines (eg, promoters inducible by interferons or interleukins).

Similarly, a variety of translational control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation start and stop codons, and picornavirus-derived elements (in particular, an internal ribosome entry site, or IRES, also referred to as IRES). CITE).

In other disclosures, a polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA).

The coding regions of polynucleotides and nucleic acids may be associated with additional coding regions encoding secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide as disclosed herein, for example, a polynucleotide encoding an antibody. or antigen-binding fragment, or a variant or derivative thereof that specifically binds Siglec-15. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once export of the growing protein chain across the reticulum has begun. rough endoplasmic. Those of skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the entire or "full-length" polypeptide to produce a secreted or "full-length" form. mature" of the polypeptide. In certain disclosures, the native signal peptide, eg, an immunoglobulin heavy chain or light chain signal peptide, or a functional derivative of that sequence that retains the ability to direct secretion of the polypeptide that is operatively associated with it, is used. . Alternatively, a heterologous mammalian signal peptide or functional derivative thereof may be used. For example, the wild-type leader sequence can be replaced by the leader sequence of human tissue plasminogen activator (TPA) or mouse p-glucuronidase.

Certain antibodies, or antigen-binding fragments, variants, analogs, or derivatives thereof, including genetically engineered antibody molecules or fragments that bind antigen in a manner similar to the disclosed antibody molecules, are disclosed herein. Also disclosed are immunoconjugates comprising said antibodies, or antigen-binding fragments, variants, analogs, or derivatives thereof.

The terms "antibody" and "immunoglobulin" may be used interchangeably herein. An antibody (or a fragment, variant or derivative thereof as disclosed herein comprises at least one heavy chain variable domain and at least one heavy chain and one light chain variable domains. Structures of antibody are relatively well known. basic immunoglobulins in vertebrate systems See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition, 1988).

As will be discussed in more detail below, the term "immunoglobulin" encompasses several broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (y, |J, a, 5, £) with some subclasses in between (eg, Y1-Y4). The nature of this chain is what determines the "class" of the antibody as IgG, IgM, IgA, IgG or IgE, respectively.

Immunoglobulin subclasses (isotypes), eg, IgGi, IgG2, IgG3, IgG4, IgAi, etc., are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible to the skilled person in view of the present disclosure, and accordingly fall within the scope of the present disclosure.

^{Light chains are classified as kappa or lambda} (k, ^{A). Each kind of heavy chain can be attached to either a kappa or a lambda light chain} . In general, the light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or noncovalent bonds when immunoglobulins are generated by hybridomas, B lymphocytes, or cells. genetically engineered hosts. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to a C-terminus at the bottom of each chain.

Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. In contrast, the light chain (CL) and heavy chain (CH1, CH2, or CH3) constant domains confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and Similar. By convention, the numbering of the constant region domains increases as they move away from the antigen-binding site or the amino terminus of the antibody. The N-terminal portion is a variable region and the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy terminus of the heavy and light chain, respectively.

As noted above, the variable region enables the antibody or antigen-binding fragment to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and the VH domain, or the complementarity determining region (CDR) subset, of an antibody or antigen-binding fragment combine to form the variable region that defines a three-dimensional antigen binding site. This quaternary structure of the antibody or antigen-binding fragment forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs on each of the VH chains. and VL.

In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen-binding domain are short, noncontiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain when the Antibody assumes its three-dimensional configuration in an aqueous environment. The rest of the amino acids in the antigen-binding domains, called "framework" regions, show less intermolecular variability. The framework regions largely adopt a p-sheet conformation and the CDRs form loops that connect to, and in some cases form part of, the p-sheet structure. Thus, the framework regions act to form a scaffold that provides for the positioning of the CDRs in the correct orientation through non-covalent inter-strand interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids that comprise the CDRs and framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined (see, "Sequences of Proteins of Immunological Interest", Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987) ,

In the event that there are two or more definitions of a term that are used and/or accepted in the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated otherwise. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the noncontiguous antigen combining sites found within the variable region of both long-chain polypeptides. heavy as light. This particular region has been described by Kabat et al., US Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), where the definitions include overlaps or subsets of amino acid residues when compared to each other. However, the application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. Suitable amino acid residues encompassing CDRs as defined in each of the references cited above are set forth in Table A below for comparison. The exact number of residues encompassed by a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the amino acid sequence of the variable region of the antibody.

TABLE A: CDR1 Definitions

Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One skilled in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence, without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., US Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983) . Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody or antigen-binding fragment, or variant or derivative thereof that specifically binds Siglec-15 as disclosed herein document are according to the Kabat numbering system.

Antibodies or antigen-binding fragments, variants, or derivatives thereof include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies, single-chain antibodies, epitope-binding fragments, eg, Fab, Fab', and F (ab')2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-linked Fv (sdFv), fragments comprising a VL or VH domain, fragments produced by a Fab expression library. ScFv molecules are known in the art and are described, for example, in US Pat.

5,892,019. Immunoglobulin or antibody molecules encompassed by this disclosure may be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass of immunoglobulin molecule.

By "binds specifically", it is generally meant that an antibody or fragment, variant or derivative thereof binds to an epitope through its antigen-binding domain, and that binding implies some complementarity between the antigen-binding domain and the epitope. In accordance with the present definition, an antibody or fragment, variant or derivative thereof is said to "specifically bind" an epitope when it binds to that epitope, via its antigen-binding domain, more readily than it otherwise would. would bind to a random unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a given antibody or fragment, variant or derivative thereof binds to a given epitope. For example, antibody 'A' can be considered to have a higher specificity for a given epitope than antibody 'B', or antibody 'A' can be said to bind epitope 'C' with a higher specificity than it does. for the related epitope "D".

By "binds preferentially" is meant that the antibody specifically binds an epitope more readily than it would bind a related, similar, homologous, or analogous epitope. Therefore, an antibody that "preferentially binds" to a given epitope would more likely bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.

An antibody or fragment, variant or derivative thereof, or an immunoconjugate comprising an antibody or fragment, variant or derivative thereof, is said to competitively inhibit the binding of a reference antibody or antigen-binding fragment to a given epitope if preferentially binds to that epitope to the extent that it blocks, to some degree, the binding of the reference antibody or antigen-binding fragment to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competitive ELISA assays. An antibody or fragment, variant or derivative thereof can be said to competitively inhibit binding of the reference antibody or antigen-binding fragment to a given epitope by at least 90%, at least 80%, at least 70% , at least 60% or at least 50%.

Antibodies or antigen-binding fragments, variants, or derivatives thereof as disclosed herein may also be described or specified in terms of their cross-reactivity. As used herein, the term "cross-reactivity" refers to the ability of an antibody or fragment, variant or derivative thereof, specific for one antigen, to react with a second antigen; a measure of the relationship between two different antigenic substances. Thus, an antibody is cross-reactive if it binds to an epitope other than the one that induced its formation. The cross-reactive epitope usually contains many of the same complementary structural features as the

Antibody fragments including single chain antibodies may comprise the variable region(s) alone or in combination with all or a portion of the following: hinge region, CH1, CH2 and CH3 domains. Also included are antibodies or antigen-binding fragments that also comprise any combination of variable region(s) with a hinge region, CH1, cH2, and CH3 domains. Antibodies or binding fragments antigen thereof disclosed herein may be of any animal origin, including birds and mammals. The antibodies can be human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibodies. In another disclosure, the variable region may be of chondrichtoid (eg, shark) origin. As used herein, "human" antibodies include antibodies that have the amino acid sequence of a human immunoglobulin and include antibodies isolated from human or animal immunoglobulin libraries that are transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins. , as described, for example, in Pat. US Patent No. 5,939,598 to Kucherlapati et al.

As used herein, the term "heavy chain portion" includes amino acid sequences derived from an immunoglobulin heavy chain. An antibody or fragment, variant, or derivative thereof, comprising a heavy chain portion comprises at least one of the following: a CH1 domain, a hinge domain (eg, upper, middle, and/or lower hinge region), a CH2, a CH3 domain, or a variant or fragment thereof. For example, an antibody or fragment, variant or derivative thereof may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, disclosure, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain . In another disclosure, an antibody or fragment, variant or derivative thereof comprises a polypeptide chain comprising a CH3 domain. In addition, an antibody or fragment, variant, or derivative thereof for use in the disclosure may lack at least a portion of a CH2 domain (eg, all or part of a CH2 domain). As discussed above, one skilled in the art will understand that these domains (eg, heavy chain portions) can be modified so as to vary in the amino acid sequence of the naturally occurring immunoglobulin molecule.

The heavy chain portions of an antibody or a fragment, variant, or derivative thereof as disclosed herein may be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain portion may comprise a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, a heavy chain portion may comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

As used herein, the term "light chain portion" includes amino acid sequences derived from an immunoglobulin light chain. The light chain portion comprises at least one of the VL or CL domains.

The antibodies or antigen-binding fragments, variants, or derivatives thereof disclosed herein may be described or specified in terms of the epitope(s), or portion(s), of an antigen that they specifically recognize or bind. The portion of a target antigen that specifically interacts with the antigen-binding domain of an antibody is an "epitope" or "antigenic determinant." A target antigen, eg, a polysaccharide, may comprise a single epitope, but typically comprises at least two epitopes and may include any number of epitopes, depending on the size, conformation, and type of antigen.

As indicated above, the subunit structures and three-dimensional configuration of the constant regions of the various classes of immunoglobulins are well known. As used herein, the term "VH domain" includes the amino terminal variable domain of an immunoglobulin heavy chain and the term "CH1 domain" includes the first (most amino terminal) constant region domain of a heavy chain. of immunoglobulin. The CH1 domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.

As used herein, the term "CH2 domain" includes the portion of a heavy chain molecule that extends, for example, from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system, and residues 231-340, EU numbering system, see Kabat EA et al. , op.cit. The CH2 domain is unique in that it is not closely paired with another domain. Rather, between the two CH2 domains of an intact native IgG molecule interpose two N-linked branched carbohydrate chains. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 remains.

As used herein, the term "chimeric antibody" means any antibody in which the immunoreactive region or site is derived from or derived from a first species and the constant region (which may be intact, partial, or modified) is derived from of a second kind. In some disclosures, the target binding region or site will be from a non-human source (eg, mouse or primate) and the constant region is human.

As used herein, the term "genetically engineered antibody" refers to an antibody in which the variable domain in the heavy and light chain or both is altered by replacement of at least partial, from one or more CDRs of an antibody of known specificity and, if necessary, by partial replacement of the framework region and change of sequence. Although the CDRs may be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisioned that the CDRs are derived from an antibody of a different class and preferably from an antibody of a different species. An engineered antibody in which one or more "donor" CDRs from a non-human antibody of known specificity are grafted onto a human heavy or light chain framework region is referred to herein as a "humanized antibody." It may not be necessary to replace all of the CDRs with the entire CDRs of the donor variable region to transfer antigen-binding ability from one variable domain to another. Rather, it may only be necessary to transfer those residues that are necessary to maintain the activity of the target binding site. Given the explanations set forth, for example, in Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, will be well within the purview of those skilled in the art, whether by performing routine experimentation or by trial and error testing. error to obtain a functional genetically engineered or humanized antibody.

As used herein, the term "genetically engineered" includes the manipulation of nucleic acid molecules or polypeptides by synthetic means (eg, by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides). or some combination of these techniques).

The terms "fusion protein" or "chimeric protein" or descriptions of a protein or polypeptide comprising two residues that are "fused" refer to a first amino acid sequence joined to a second amino acid sequence with which it is not linked. naturally bound in nature. The amino acid sequences may exist normally in separate proteins that are brought together in the fusion polypeptide or may exist normally in the same protein but are placed in a new arrangement in the fusion polypeptide. A fusion protein can be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired ratio. In this document, the terms "joined", "merged" or "fusion" are used interchangeably.

The term "immunoconjugate" or "conjugate," as used herein, refers to a compound or a derivative thereof that is fused to, or bound to, a cell-binding agent (i.e., an anti-antibody). Siglec-15 or fragment thereof) and is defined by a generic formula: C-L-A, where C=cytotoxin, L=linker, and A=cell binding agent or anti-Siglec-15 antibody or antibody fragment. Immunoconjugates can also be defined by the generic formula in reverse order: A-L-C. An "immunoconjugate" comprises a targeting moiety, or moiety, such as an antibody or fragment thereof that retains antigen recognition ability, and an effector molecule, such as a therapeutic moiety or detectable label.

An "immunotoxin" is an immunoconjugate in which the therapeutic moiety is a cytotoxin. A "targeting moiety" is that portion of an immunoconjugate intended to target the immunoconjugate to a cell of interest, eg, an anti-Siglec-15 antibody or an antigen-binding fragment thereof. Typically, the targeting moiety is an antibody, scFv, dsFv, Fab, or F(ab')2. The targeting moiety may also comprise, for example, a Fab', an Fd, a V-NAR domain, an IgNar, an intrabody, an IgGACH2, a minibody, an F(ab')3, a tetrabody, a tribody, a diabody, a single domain antibody, DVD-Ig, Fcab, mAb2, a (scFv)2 or a scFv-Fc.

A "toxic moiety" is that portion of an immunotoxin that renders the immunotoxin cytotoxic to cells of interest.

A "therapeutic moiety" is that portion of an immunoconjugate intended to act as a therapeutic agent and, in some disclosures, may be a "toxic moiety."

The term "expression" as used herein refers to a process by which a gene produces a biochemical substance, eg, a polypeptide. The process includes any manifestation of the gene's functional presence within the cell, including, without limitation, gene inactivation, as well as both transient expression and stable expression. It includes, without limitation, the transcription of the gene into messenger RNA (mRNA) and the translation of said mRNA into one or more polypeptides. In case the desired end product is a biochemical agent, the term includes the creation of said biochemical agent and any precursors. The expression of a gene produces a "gene product". As used herein, a gene product can be a nucleic acid, eg, a messenger RNA produced by transcription of a gene, or a polypeptide that is translated from a transcript. The gene products described herein further include nucleic acids with post-transcriptional modifications, eg, polyadenylation, or polypeptides with post-translational modifications, eg, methylation, glycosylation, lipid addition, association with other protein subunits, proteolytic cleavage, and the like.

As used herein, the terms "treat" or "treatment" refer to therapeutic measures, where the goal is to prevent or slow down (lessen) an undesired physiological change or disorder. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, decreased extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression , improvement or palliation of disease and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to expected survival if no treatment is received.

By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, particularly a mammalian subject, for whom a diagnosis, prognosis or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo animals, sporting animals, or companion animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears. , etc.

II. ANTIBODIES OR ANTIGEN-BINDING FRAGMENTS

Siglec-15 is a type I transmembrane protein comprising two immunoglobulin-like domains, a transmembrane domain containing a lysine residue, and a short cytoplasmic tail. The extracellular domain of Siglec-15 preferentially recognizes Neu5Aca2-6GalNAca structures and has immune system activating activity. Although previously identified on dendritic cells and macrophages, Siglec-15 has been shown to be expressed at high levels on the surface of AML blasts and at low levels on peripheral blood leukocytes.

One disclosure relates to an antibody or antigen-binding fragment thereof that specifically binds Siglec-15. In certain disclosures, the antibody or antigen-binding fragment thereof is A9E8. In other disclosures, the antibody or antigen-binding fragment thereof is an immunoconjugate comprising an antibody against Siglec-15 or an antigen-binding fragment such as A9E8. The disclosure further relates to an antibody or antigen-binding fragment thereof that specifically binds to the same epitope of Siglec-15 as an antibody or antigen-binding fragment thereof comprising the heavy chain variable region (VH) and the disclosed light chain variable region (VL) of A9E8. Further disclosed is an antibody or antigen-binding fragment thereof that specifically binds to Siglec-15 and competitively inhibits binding to Siglec-15 by an antibody or antigen-binding fragment thereof comprising the VH and/or VL of A9E8; the A9E8 VH-CDR3 and/or the A9E8 VL-CDR3; the VH-CDR1, VH-CDR2 and VH-CDR3 of A9E8; the VL-CDR1, VL-CDR2 and VL-CDR3 of A9E8; or the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of A9E8. An antibody is said to competitively inhibit the binding of a reference antibody to a given epitope if it binds preferentially to that epitope to the point of blocking, to some degree, the reference antibody's binding to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competitive ELISA assays. An antibody can be said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

Due to the differential expression of Siglec-15 in AML blasts compared to normal peripheral blood leukocytes, Siglec-15 can be used to screen for additional therapies, for example, antibodies that bind to Siglec-15 and immunoconjugates comprising Siglec-15. anti-Siglec-15 antibodies, which preferentially target AML blasts and have minimal effects on normal peripheral blood leukocytes. In one disclosure, the Siglec-15 therapy is an anti-Siglec-15 antibody or antigen-binding fragment. In another disclosure, the anti-Siglec-15 therapy is an immunoconjugate comprising an anti-Siglec-15 targeting moiety. In a further disclosure, the Siglec-15 therapy is a small molecule.

Methods of making antibodies are well known in the art and are described herein. Once antibodies have been produced against various Siglec-15 fragments, or against the full length of it, without the signal sequence, determining which amino acids, or epitope, of Siglec-15 to which the antibody or fragment binds Antigen binding activity can be determined by epitope mapping protocols, as described herein, as well as by methods known in the art (e.g., double-antibody sandwich ELISA, as described in "Chapter 11 - "Immunology"). , in "Current Protocols in Molecular Biology", Ed. Ausubel et al., v.2, John Wiley & Sons, Inc. (1996). Additional epitope mapping protocols can be found in Morris, G. Epitope Mapping Protocols, New Jersey: Humana Press (1996) Epitope mapping can also be done by commercial means (ie, ProtoPROBE, Inc. (Milwaukee, Wisconsin)).

In certain aspects, the disclosure relates to an antibody or fragment, variant, derivative, or immunoconjugate thereof that specifically binds to Siglec-15 and induces an enhanced rate of Siglec-15 endocytosis. In certain disclosures, an antibody or fragment, variant, derivative, or immunoconjugate thereof of the invention induces endocytosis of Siglec-15 at a rate at which the half-life of surface endogenous Siglec-15 on K562 human myelogenous leukemia cells is lower. to about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, about 1 minute, or about 30 seconds.

Unless specifically noted, as used herein, a "fragment thereof" in reference to an antibody refers to an antigen-binding fragment, ie, a portion of the antibody that specifically binds antigen.

Accordingly, disclosed herein is an anti-Siglect-15 antibody or antigen-binding fragment, variant or derivative thereof, wherein at least a fraction of one or more of the constant region domains has been deleted. or otherwise altered to provide desired biochemical characteristics, such as reduced effector functions, non-covalent dimerization ability, increased localization ability at the site of a tumor, a reduced serum half-life or an increased serum half-life compared to a complete and unchanged antibody of approximately the same immunogenicity. For example, certain antibodies or antigen-binding fragments thereof described herein are antibodies with deletion domains that comprise a polypeptide chain similar to an immunoglobulin heavy chain, but that lack at least a portion of one or more heavy chain domains. For example, in certain antibodies, an entire constant region domain of the modified antibody will be deleted, eg, all or part of the CH2 domain will be deleted.

Modified forms of anti-Siglec-15 antibodies or antigen-binding fragments, variants, or derivatives thereof can be made from whole precursor or parent antibodies using techniques known in the art. Example techniques are discussed elsewhere in this document.

In certain disclosures, both the variable and constant regions of anti-Siglect-15 antibodies or antigen-binding fragments are fully human. Fully human antibodies can be made using techniques that are known in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been knocked out. Exemplary techniques that can be used to make such antibodies are described in US Patents: 6,150,584; 6,458,592; 6,420,140. Other techniques are known in the art. Fully human antibodies can also be produced by various display technologies, eg, phage display or other viral display systems, as described in more detail herein.

Anti-Siglec-15 antibodies or antigen-binding fragments thereof as disclosed herein can be prepared or manufactured using techniques known in the art. In certain disclosures, antibodies or fragments thereof are "recombinantly produced," that is, they are produced using recombinant DNA technology. Exemplary techniques for making antibody molecules or fragments thereof are discussed in more detail herein.

In certain anti-Siglec-15 antibodies, the Fc portion can be mutated to decrease effector function using techniques known in the art. For example, deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of circulating modified antibody, thereby increasing tumor localization. In other cases, it may be that constant region modifications moderate complement binding and thus reduce the serum half-life and nonspecific association of a conjugated cytotoxin. However, other constant region modifications can be used to modify disulfide bonds or oligosaccharide moieties that allow for enhanced localization due to greater antigen specificity or antibody flexibility. The physiological profile, bioavailability, and other biochemical effects resulting from the modifications, such as localization, biodistribution, and serum half-life, can be readily measured and quantified using well-known immunological techniques without the need for undue experimentation.

In certain disclosures, anti-Siglec-15 antibodies or antigen-binding fragments, variants, immunoconjugates, or derivatives thereof will not elicit a deleterious immune response in the animal to be treated, eg, a human. In one disclosure, anti-Siglec-15 antibodies or antigen-binding fragments, variants, immunoconjugates, or derivatives thereof are modified to reduce their immunogenicity using art-recognized techniques. For example, antibodies can be humanized, deimmunized, or chimeric antibodies can be made. These types of antibodies are derived from a non-human antibody, typically a murine or primate antibody, that retains or substantially retains the antigen-binding properties of the parent antibody, but is less immunogenic in humans. This can be achieved by a variety of methods, including (a) grafting the entire non-human variable domains onto human constant regions to generate chimeric antibodies; (b) grafting at least a portion of one or more non-human complementarity determining regions (CDRs) onto a human framework and constant regions with or without retention of critical framework residues; or (c) transplanting the entire non-human variable domains, but "cloaking" them with a human-like section by surface residue replacement. Such methods are disclosed in Morrison et al., Proc. Natl. Acad. Sci. 81:6851-6855 (1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 28:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994) and Pat. US Nos. 5,585,089, 5,693,761, 5,693,762 and 6,190,370,

Deimmunization can also be used to decrease the immunogenicity of an antibody. As used herein, the term "deimmunization" includes alteration of an antibody to modify T cell epitopes (see, eg, WO9852976A1, WO0034317A2). For example, the VH and VL sequences of the starting antibody are analyzed and a "map" of human T cell epitopes of each V region is obtained showing the location of the epitopes with respect to the complementarity determining regions (CDRs). ) and other key residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering the activity of the final antibody. A series of alternative VH and VL sequences comprising combinations of amino acid substitutions are designed and these sequences are subsequently incorporated into a series of binding polypeptides, eg, Siglec-15-specific antibodies or antigen-binding fragments thereof. disclosed herein, the function of which is then tested. Complete heavy and light chain genes comprising modified human V and C regions are then cloned into expression vectors and subsequent plasmids introduced into cell lines for production of the complete antibody. The antibodies are then compared in suitable biochemical and biological assays and the optimal variant is identified.

Anti-Siglec-15 antibodies or antigen-binding fragments, variants, immunoconjugates or derivatives thereof can be generated by any suitable method known in the art. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques, including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988)

DNA encoding antibodies or antibody fragments (eg, antigen binding sites) can also be derived from antibody libraries, such as phage display libraries. In particular, such phage can be used to display antigen-binding domains expressed from a combinatorial antibody repertoire or library (eg, human or murine). Phage displaying an antigen-binding domain that binds to the antigen of interest can be selected or antigen-identified, for example, using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with scFv, Fab, Fv OE dAb (individual Fv heavy or light chain region) or disulfide stabilized Fv antibody domains. recombinantly fused with gene III or gene VIII protein of phage. Exemplary methods are set forth, for example, in EP 368684 B1; US Patent 5,969,108, Hoogenboom, H.R. and Chames, Immunol. Today 21:371 (2000); Nagy et al. Nat. Med. 8:801 (2002); Huie et al., Proc. Natl. Acad. Sci. USA 98:2682 (2001); Lui et al., J. Mol. Biol.

315:1063 (2002). Several publications (eg, Marks et al., BiolTechnology 10:779-783 (1992)) have described the production of high-affinity human antibodies by chain exchange, as well as combinatorial infection and in vivo recombination as a strategy to construct large phage libraries. In another disclosure, ribosomal display can be used to replace bacteriophage as the display platform (see, eg, Hanes et al., Nat. Biotechnol. 18:1287 (2000); Wilson et al., Proc. Natl. Acad. Sci USA 98:3750 (2001) or Irving et al., J. Immunol.Methods 248:31 (2001)). In another disclosure, cell surface libraries can be screened for antibodies (Boder et al., Proc. Natl. Acad. Sci. USA 97:10701 (2000); Daugherty et al., J. Immunol. Methods 243: 211 (2000)). Such procedures provide alternatives to traditional hybridoma techniques for the isolation and subsequent cloning of monoclonal antibodies.

In phage display methods, functional antibody domains are displayed on the surface of phage particles bearing the polynucleotide sequences that encode them. For example, the DNA sequences encoding the VH and VL regions are amplified from animal cDNA libraries (eg, human or murine cDNA libraries from lymphoid tissues) or synthetic cDNA libraries. In certain disclosures, the DNA encoding the VH and VL regions is linked by an scFv linker by PCR and cloned into a phagemic vector (eg, pCANTAB 6 or pComb 3 HSS). The vector is electroporated into E. coli and the E. coli is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and M13, and the VH or VL regions are typically recombinantly fused to gene III or gene VIII of the phage. Phage displaying an antigen-binding domain that binds an antigen of interest can be selected or antigen-identified, for example, using labeled antigen or antigen bound or captured to a solid surface or bead.

Additional examples of phage display methods that can be used to make the antibodies include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187:9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT Application No. p Ct /GB91/01134; Pc T publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and the Pats. US Patent No. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;

As described in the references above and in the examples below, after phage selection, the antibody coding regions of the phage can be isolated and used to generate whole antibodies, including human antibodies or any other desired antigen-binding fragments. and can be expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques for recombinantly producing Fab, Fab', and F(ab') 2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988). Examples of techniques that can be used to produce single-chain Fv and antibodies include those described in US Pat. US Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988).

Fully human antibodies are particularly desirable for the therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art, including phage display methods described above, using antibody libraries derived from sequences of human immunoglobulins. See also Pat. US Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741.

Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins, but can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. In addition, various companies may be contracted to provide human antibodies produced in transgenic mice directed against a selected antigen using technology similar to that described above.

Fully human antibodies that recognize a selected epitope can be generated using a technique called "guided selection." In this approach, a selected non-human monoclonal antibody, eg, a mouse antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope. (Jespers et al., Bio/Technology 12:899-903 (1988). See also, US Patent No. 5,565,332).

In another disclosure, DNA encoding the desired monoclonal antibodies can be readily isolated and sequenced using conventional methods (eg, by use of oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). . Isolated and subcloned hybridoma cells or isolated phage, for example, can serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, English) or myeloma cells that do not otherwise produce immunoglobulins. More particularly, the isolated DNA (which may be synthetic as described herein) can be used to clone constant and variable region sequences for antibody manufacture as described in Newman et al., Pat. No. 5,658,570, filed January 25, 1995. Transformed cells expressing the desired antibody can be cultured in relatively large quantities to provide clinical and commercial supplies of the immunoglobulin.

In one disclosure, an isolated antibody or antigen-binding fragment thereof comprises at least one heavy or light chain CDR of an antibody molecule, eg, A9E8. In another disclosure, an isolated antibody or antigen-binding fragment thereof comprises at least two CDR disclosures of one or more antibody molecules, eg, A9E8. In another disclosure, an isolated antibody or antigen-binding fragment thereof comprises at least three CDRs from one or more antibody molecules, eg, A9E8. In another disclosure, an isolated antibody or antigen-binding fragment thereof comprises at least four CDRs from one or more antibody molecules, eg, A9E8. In another disclosure, an isolated antibody or antigen-binding fragment thereof comprises at least five CDRs from one or more antibody molecules, eg, A9E8. In another disclosure, an isolated antibody or antigen-binding fragment thereof of the disclosure comprises at least six CDRs from one or more antibody molecules, eg, A9E8.

In a specific disclosure, the amino acid sequence of the heavy and/or light chain variable domains can be inspected to identify the complementarity determining region (CDR) sequences by methods well known in the art, for example, by comparison with known amino acid sequences of other heavy and light chain variable regions to determine regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs can be inserted into framework regions, eg, into human framework regions to humanize a non-human antibody. Framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, eg, Chothia et al., J. Mol. Biol. 278:457-479 (1998) for a listing of human framework regions). The polynucleotide generated by combining the framework regions and CDRs encodes an antibody that specifically binds to at least one epitope of a desired antigen, eg, Siglec-15. One or more amino acid substitutions can be made within the framework regions and the amino acid substitutions improve the binding of the antibody to its antigen. In addition, such methods can be used to make amino acid substitutions or deletions of one or more variable region cysteine residues that participate in an intrachain disulfide bond to generate antibody molecules that lack one or more intrachain disulfide bonds. Other alterations of the polynucleotide are encompassed by the present disclosure and are within the capabilities of one of ordinary skill in the art.

Also disclosed are antibodies or antigen-binding fragments thereof that comprise, consist essentially of, or consist of variants (including derivatives) of antibody molecules (eg, VH regions and/or VL regions) described in the herein, which antibodies or antigen-binding fragments specifically bind Siglec-15. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding an antibody or antigen-binding fragment that specifically binds to Siglec-15, including, without limitation, site-directed mutagenesis and mutagenesis. PCR-mediated resulting in amino acid substitutions. Variants (including derivatives) encode polypeptides comprising fewer than 50 amino acid substitutions, fewer than 40 amino acid substitutions, fewer than 30 amino acid substitutions, fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions of amino acids, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference VH region, VHCDR1, VHCDR2, VHCDR3, the VL region, VLCDR1, VLCDR2, or VLCDR3. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similarly charged side chain. Families of amino acid residues having similarly charged side chains have been defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic (eg, tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations may be introduced randomly throughout all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants screened for biological activity to identify mutants that retain activity (e.g., the ability to bind to Siglec-15).

For example, it is possible to introduce mutations only in the framework regions or only in the CDR regions of an antibody molecule. The introduced mutations may be silent or neutral missense mutations, ie they have little or no effect on the ability of an antibody to bind antigen. These types of mutations may be useful for optimizing codon usage or improving the production of a hybridoma antibody. Alternatively, non-neutral missense mutations can alter an antibody's ability to bind antigen. The location of most silent and neutral missense mutations is likely to be in the framework regions, while the location of most non-neutral missense mutations is likely to be in a CDR. , although this is not an absolute requirement. One of skill in the art would be able to design and test mutant molecules with desired properties, such as no alteration of antigen binding activity or alteration of binding activity (e.g. enhancements in binding activity). to antigen or changes in antibody specificity). After mutagenesis, the encoded protein can be routinely expressed and the functional and/or biological activity of the encoded protein, (eg, the ability to bind to at least one epitope of Siglec-15) can be determined using the techniques described. herein or by routine modification techniques known in the art.

ME. ANTIBODY POLYPEPTIDES AND IMMUNOCONJUGATES

The disclosure further relates to isolated polypeptides constituting antibodies or antigen-binding fragments thereof, which specifically bind Siglec-15, and to polynucleotides encoding such polypeptides. Isolated antibodies or fragments thereof as disclosed herein comprise polypeptides, eg, amino acid sequences encoding, eg, Siglec-15-specific antigen-binding regions derived from immunoglobulin molecules. A polypeptide or amino acid sequence "derived from" an indicated protein refers to the origin of the polypeptide. In certain instances, the polypeptide or amino acid sequence that is derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is essentially identical to that of the starting sequence, or a portion thereof, wherein the portion consists of at least 10-20 amino acids, at least 20-30 amino acids, at least 30-50 amino acids, or is otherwise identifiable by one of ordinary skill in the art as originating from the starting sequence.

Also disclosed is an isolated antibody or antigen-binding fragment thereof that specifically binds Siglec-15 and that comprises an immunoglobulin heavy chain (VH) variable region amino acid sequence that is at least 80%, 85 %, 90% 95% or 100% identical to SEQ ID NO: 1 as shown in Table 1.

Furthermore, an isolated antibody or antigen-binding fragment thereof that specifically binds to Siglec-15 and that comprises an amino acid sequence of VH identical to, or identical except for one, two, three, four, five or more is disclosed. amino acid substitutions to SEQ ID NO: 1 as shown in Table 1.

Also disclosed is an isolated antibody or antigen-binding fragment thereof that specifically binds Siglec-15 and that comprises an immunoglobulin light chain (VL) variable region amino acid sequence that is at least 80%, 85 %, 90% 95% or 100% identical to SEQ ID NO: 2 as shown in Table 1.

Also disclosed is an isolated antibody or antigen-binding fragment thereof that specifically binds Siglec-15 and that comprises a VL amino acid sequence identical to, or identical except for one, two, three, four, five or more substitutions. of amino acids to SEQ ID NO: 2 as shown in Table 1.

Also disclosed is an isolated antibody or antigen-binding fragment thereof that specifically binds to Siglec-15 comprising a VH-CDR1, VH-CDR2 and/or VH-CDR3, where the VH-CDR1, VH-CDR2 and VH -CDR3 correspond to SEQ ID NO: 3, SEQ ID NO: 4 and s Eq ID NO: 5, respectively. In further disclosures, the isolated antibody or antigen-binding fragment thereof comprises a VL-CDR1, VL-CDR2 and/or VL-CDR3, where VL-CDR1, VL-CDR2 and VL-CDR3 correspond to SEQ ID NO : 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively. In particular disclosures, the isolated antibody or antigen-binding fragment thereof comprises a VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and/or VL-CDR3, where the VH-CDR1, VH-CDR1, CDR2 VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 correspond to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

Also disclosed is an isolated antibody or antigen-binding fragment thereof that specifically binds to Siglec-15 comprising a VH-CDR1, VH-CDR2 and/or VH-CDR3; a VL-CDR1, VL-CDR2 and/or VL-CDR3; or a VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and/or VL-CDR3; wherein the sequence of each CDR is identical, or identical except for one, two or three amino acid substitutions in each CDR with respect to the sequences of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 corresponding to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

In certain disclosures, an isolated antibody or antigen-binding fragment thereof as described herein specifically binds Siglec-15 and causes an increased rate of endocytosis of the receptor. In some disclosures, the half-life of Siglec-15 endocytosis is less than about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, about 1 minute, or about 30 seconds in the human myelogenous leukemia cell line K562.

TABLE 1: Reference VH and VL amino acid sequences

VH name VH-CDR1 antibody

A9E8 NWWS (SE QLQLQESGPGLVKPSET NO:3) EVHHSGVTEFADDAFD

Any anti-Siglec-15 antibodies or fragments, variants, or derivatives thereof described herein may further include additional polypeptides, for example, a signal peptide to direct secretion of the encoded polypeptide, antibody constant regions as described in herein or other heterologous polypeptides as described herein. Additionally, the antibodies or fragments thereof of the disclosure include polypeptide fragments as described elsewhere. Additionally, the anti-Siglec-15 antibodies or fragments, variants, or derivatives thereof described herein may be fusion polypeptides, Fab fragments, scFvs, or other derivatives, as described herein.

Additionally, as described in more detail elsewhere herein, the disclosure includes compositions comprising anti-Siglec-15 antibodies or fragments, variants, or derivatives thereof described herein.

One of skill in the art will also understand that the anti-Siglec-15 antibodies or fragments, variants, or derivatives thereof described herein may be modified so as to vary in the amino acid sequence of the naturally occurring binding polypeptide from which they were derived. . For example, a polypeptide or amino acid sequence derived from an indicated protein may be similar, eg, have a certain percentage identity to the starting sequence, eg, it may be 60%, 70%, 75%, 80% , 85%, 90% or 95% identical to the starting sequence.

As is known in the art, "sequence identity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide with the sequence of a second polypeptide. When discussed herein, whether any particular polypeptide is at least about 70%, 75%, 80%, 85%, 90%, or 95% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best homology segment between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, say, 95% identical to a reference sequence, the parameters are, of course, set such that percent identity is calculated over the full length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are permitted.

In addition, nucleotide or amino acid substitutions, deletions, or insertions can be made that lead to conservative substitutions or changes in "non-essential" amino acid regions. For example, a polypeptide or amino acid sequence derived from an indicated protein may be identical to the starting sequence except for one or more single amino acid substitutions, insertions, or deletions, e.g., one, two, three, four, five, six , seven, eight, nine, ten, fifteen, twenty or more single amino acid substitutions, insertions or deletions. In certain disclosures, a polypeptide or amino acid sequence derived from a recited protein has one to five, one to ten, one to fifteen, or one to twenty single amino acid substitutions, insertions, or deletions relative to the parent sequence. .

An anti-Siglec-15 antibody or fragment, variant, or derivative thereof described herein may comprise, consist essentially of, or consist of a fusion protein. Fusion proteins are chimeric molecules comprising, for example, an immunoglobulin antigen-binding domain with at least one target-binding site and at least one heterologous portion, i.e., a portion with which it is not directly bound. natural form in nature. The amino acid sequences may normally exist in separate proteins that are brought together in the fusion polypeptide or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. Fusion proteins can be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired ratio.

The term "heterologous" applied to a polynucleotide, polypeptide, or other moiety means that the polynucleotide, polypeptide, or other moiety is derived from an entity other than the moiety of the entity with which it is compared. In one non-limiting example, a "heterologous polypeptide" to be fused with an antibody or antigen-binding fragment, variant or derivative thereof is derived from a non-immunoglobulin polypeptide of the same species or an immunoglobulin or non-immunoglobulin polypeptide of the same species. immunoglobulin of a different species.

Toxins with antibodies or polypeptides of the present disclosure can be used to obtain immunoconjugates. Exemplary toxins include ricin, abrin, diphtheria toxin and subunits thereof, as well as botulinum toxins A through F. These toxins are readily available from commercial sources (eg, Sigma Chemical Company, St. , Mo.). Diphtheria toxin is isolated from Corynebacterium diphtheriae. Ricin is the lectin RCA60 from Ricinus communis (castor bean). The term also refers to toxic variants of it. See Pat. US Nos. 5,079,163 and 4,689,401. Ricinus communis agglutinin (RCA) occurs in two forms designated RCA60 and RCA120 based on their molecular weights of approximately 65,000 and 120,000, respectively. Nicholson and Blaustein, J. Biochim. Biophys. Minutes, 266:543 (1972). The A chain is responsible for the inactivation of protein synthesis and cell death. The B chain binds ricin to cell surface galactose residues and facilitates the transport of the A chain to the cytosol (Olsnes et al., Nature, 1974;249:627-631). See Pat. No. 3,060,165.

In one disclosure, the toxin is Pseudomonas exotoxin. Pseudomonas exotoxin A (PE) is an extremely active monomeric protein (molecular weight of 66 kD), secreted by Pseudomonas aeruginosa, that inhibits protein synthesis in eukaryotic cells through inactivation of exotoxin factor. elongation 2 (EF-2) by catalyzing its ADP-ribosylation (catalyzing the transfer of the ADP-ribosyl moiety from oxidized NAD to EF-2).

Pseudomonas exotoxins (PE) can include the native sequence, cytotoxic fragments of the native sequence, and conservatively modified variants of native PE and its cytotoxic fragments. Cytotoxic fragments of PE include those that are cytotoxic with or without subsequent proteolytic processing or other processing in the target cell (eg, as protein or preprotein). Cytotoxic fragments of PE include PE40, PE38, and PE35. PE40 is a truncated derivative of PE as previously described in the art. See, Pai et al., Proc. Natl. Acad. Sci. USA, 88:3358-62 (1991); Kondo et al., J. Biol. Chem. 263:9470-9475 (1988). PE38 is a truncated PE composed of amino acids 253-364 and 381-613. PE35 is a 35 kD carboxyl-terminal fragment composed of a Met at position 280 followed by amino acids 281-364 and 381-613 of native PE. In preferred disclosures, the cytotoxic fragment PE38 is used. PE38 is a proprotein that can be activated to its cytotoxic form after processing within a cell.

With the Pseudomonas exotoxins and antibodies disclosed herein, one of skill can easily construct a variety of clones containing functionally equivalent nucleic acids, such as fatty acids. nucleic acids that differ in sequence but encode the same PE or antibody sequence. Therefore, the present invention discloses nucleic acids encoding antibodies and conjugates and fusions thereof.

In other disclosures, the toxin is a holotoxin (eg, type II ribosome-inactivating protein) or a hemitoxin (eg, saporin, gelonin). In additional disclosures, toxin is a cytotoxic agent or compound, examples of which include taxanes, DNA alkylating agents, anthracyclines, microtubule inhibitors (eg, tubulisins), duocarmycins, maytansinoids, doxorubicin, and auristatins.

IV. POLYNUCLEOTIDES ENCODING ANTIBODIES OR ANTIGEN-BINDING FRAGMENTS THEREOF

Also disclosed herein are nucleic acid molecules encoding the anti-Siglec-15 antibodies or fragments, variants, or derivatives thereof described herein.

Disclosed herein is an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain (VH) variable region amino acid sequence that is at least 80%, 85%, 90 % 95% or 100% identical to SEQ ID NO: 1 as shown in Table 1. An isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an amino acid sequence is disclosed herein. of VH identical to, or identical except for one, two, three, four, five or more amino acid substitutions to SEQ ID NO: 1 as shown in Table 1.

An isolated antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide that binds specifically or preferentially to Siglec-15 is disclosed herein.

Disclosed herein is an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain (VL) variable region amino acid sequence that is at least 80%, 85%, 90% 95% or 100% identical to SEQ ID NO: 2 as shown in Table 1. An isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an amino acid sequence is disclosed herein. of VL identical to, or identical except for one, two, three, four, five or more amino acid substitutions to SEQ ID NO: 2 as shown in Table 1.

In another disclosure, an isolated antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide binds specifically or preferentially to Siglec-15.

An isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid at least 80%, 85%, 90%, 95% or 100% identical to s Eq ID NO: 9 or SEQ ID NO: 10 as shown in Table 2.

TABLE 2: Reference Nucleic Acid Sequences

In some disclosures, an isolated antibody or antigen-binding fragment thereof encoded by one or more of the polynucleotides described above, that specifically binds Siglec-15, comprises, consists essentially of, or consists of VH and VL amino acid sequences at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The disclosure also includes fragments of the polynucleotides as described elsewhere herein. Additionally, polynucleotides encoding fusion polynucleotides, Fab fragments, and other derivatives are disclosed, as is also disclosed.

The polynucleotides can be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (eg, as described in Kutmeier et al., BioTechniques 17:242 (1994)), which , briefly, involves the synthesis of overlapping oligonucleotides containing portions of the antibody-encoding sequence, hybridization and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an anti-Siglec-15 antibody or fragment, variant, or derivative thereof may be generated from nucleic acid of a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the antibody can be chemically synthesized or obtained from a suitable source (for example, a antibody cDNA library or a cDNA library generated from, or nucleic acid, preferably poly A+RNA, isolated from, any tissue or cells expressing the antibody or such as hybridoma cells selected to express an antibody) by amplification by PCR using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, for example, a cDNA clone from a cDNA library encoding the antibody . The amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence of an anti-Siglec-15 antibody or fragment, variant, or derivative thereof has been determined, its nucleotide sequence can be manipulated using methods well known in the art for nucleotide sequence manipulation. , for example, recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (See, for example, the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998), to generate antibodies having a different amino acid sequence, eg, to create amino acid substitutions, deletions, and/or insertions.

A polynucleotide encoding an anti-Siglec-15 antibody or fragment, variant or derivative thereof may be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or αDn. For example, a polynucleotide encoding an anti-Siglec-15 antibody or fragment, variant, or derivative thereof may be composed of single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single-stranded and double-stranded regions. Furthermore, a polynucleotide encoding an anti-Siglec-15 antibody or fragment, variant or derivative thereof may be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide encoding an anti-Siglec-15 antibody or fragment, variant, or derivative thereof may also contain one or more modified bases or modified DNA or RNA backbones for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA, therefore, the term "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms.

An isolated polynucleotide encoding an unnatural variant of an immunoglobulin-derived polypeptide (eg, an immunoglobulin heavy chain portion or light chain portion) can be created by introducing one or more nucleotide substitutions, additions, or deletions in the sequence of immunoglobulin nucleotides so as to introduce one or more amino acid substitutions, additions, or deletions to the encoded protein. Mutations can be introduced by conventional techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are made at one or more nonessential amino acid residues.

V. EXPRESSION OF ANTIBODY POLYPEPTIDES

As is well known, RNA can be isolated from parent hybridoma cells or other transformed cells by conventional techniques, such as extraction and guanidinium isothiocyanate precipitation followed by centrifugation or chromatography. When desired, mRNA can be isolated from total RNA by conventional techniques such as oligo dT cellulose chromatography. Suitable techniques are known in the art.

In one disclosure, cDNAs encoding the heavy and light chains of the anti-Siglec-15 antibody or fragment, variant, or derivative thereof can be made, either simultaneously or separately, using reverse transcriptase and DNA polymerase according to well-known methods. . PCR can be initiated with consensus constant region primers or with more specific primers based on published heavy and light chain DNA and amino acid sequences. As discussed above, PCR can also be used to isolate DNA clones encoding the heavy and light chains of the antibody. In this case, libraries can be screened using consensus primers or larger homologous probes, such as mouse constant region probes.

The DNA, typically plasmid DNA, can be isolated from cells using techniques known in the art, restriction mapped and sequenced according to conventional and well-known techniques set forth in detail, for example, in the above references relating to recombinant DNA techniques. Of course, the DNA may be synthetic in accordance with the present disclosure at any point in the isolation process or subsequent analysis.

Following manipulation of the isolated genetic material to provide an anti-Siglec-15 antibody or fragment, variant, or derivative thereof of the disclosure, polynucleotides encoding anti-Siglec-15 antibodies or antigen-binding fragments thereof are inserted usually in an expression vector for introduction into host cells which can be used to produce the desired amount of anti-Siglec-15 antibodies or antigen-binding fragments thereof.

Recombinant expression of an antibody or fragment, derivative or analog thereof, eg, an antibody heavy or light chain that binds to a target molecule described herein, eg, Siglec-15, requires the construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule or an antibody heavy or light chain or portion thereof (containing the heavy or light chain variable domain) of the disclosure has been obtained, the vector for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. Therefore, methods for preparing a protein by expressing a polynucleotide containing a nucleotide sequence encoding the antibody are described herein. Methods well known to those of skill in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, in vivo genetic synthesis and recombination techniques. Replicable vectors are disclosed herein that comprise a nucleotide sequence encoding an antibody molecule of the disclosure, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. . Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, eg, PCT Publication WO 86/05807; PCT Publication WO 89/01036; and US Pat. No. 5,122,464) and the variable domain of the antibody can be cloned into such a vector for expression of the entire heavy or light chain.

The term "vector" or the term "expression vector" is used herein to refer to vectors used in accordance with the present disclosure as a vehicle for introducing and expressing a desired gene in a host cell. As those skilled in the art know, such vectors can be readily selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the present disclosure will comprise a selection marker, suitable restriction sites to facilitate cloning of the desired gene, and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.

For purposes of the present disclosure, numerous expression vector systems may be employed. For example, one class of vector uses DNA elements that are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV, or MOMLV), or SV40 viruses. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells that have integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. The marker can provide prototrophy to a auxotrophic host, resistance to biocides (eg antibiotics) or resistance to heavy metals such as copper. The selectable marker gene may be directly linked to the DNA sequences to be expressed or introduced into the same cell by cotransformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals.

In some disclosures, cloned variable region genes are inserted into an expression vector along with synthetic (eg, human) heavy and light chain constant region genes as discussed above. Of course, any expression vector that is capable of triggering expression in eukaryotic cells can be used in the present disclosure. Examples of suitable vectors include, but are not limited to, plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1 and pZeoSV2 (available from Invitrogen, San Diego, CA), and plasmid pCI (available from Promega, Madison, WI). In general, screening large numbers of transformed cells for those expressing suitably high levels of immunoglobulin heavy and light chains is routine experimentation that can be performed, for example, by robotic systems.

More generally, once the vector or DNA sequence encoding a monomeric subunit of the anti-Siglec-15 antibody or fragment, variant, or derivative thereof of the disclosure has been prepared, the expression vector can be introduced into a suitable host cell. Introduction of the plasmid into the host cell can be accomplished by a variety of techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, DNA-enveloped cell fusion, microinjection, and infection with intact virus. See, Ridgway, A. A. G. "Mammalian Expression Vectors" Vectors, Rodriguez and Denhardt, Eds., Butterworths, Boston, Mass., Chapter 24.2, pgs. 470-472 (1988). Typically, introduction of the plasmid into the host is via electroporation. Host cells harboring the expression construct are cultured under conditions suitable for heavy and light chain production, and assayed for heavy and/or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or fluorescence-activated cell sorter (FACS), immunohistochemistry, and the like.

The expression vector is transferred into a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody for use in the methods described herein. Thus, the disclosure includes host cells that contain a polynucleotide encoding the anti-Siglec-15 antibody or fragment, variant, or derivative thereof, or a heavy or light chain thereof, operably linked to a heterologous promoter. In some disclosures for expression of double-chain antibodies, vectors encoding both heavy and light chains can be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

Certain disclosures include an isolated polynucleotide comprising a nucleic acid encoding the VH and VL described above, wherein an antibody or antigen-binding fragment expressed by the polynucleotide specifically binds Siglec-15. In some disclosures, the disclosed polynucleotide encodes an scFv molecule including VH and VL that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 4 as shown in Table 2. Disclosures

Some disclosures include vectors comprising the polynucleotides described above. In other disclosures, polynucleotides are operatively associated with a promoter. Host cells comprising such vectors are disclosed herein. Also disclosed herein are vectors in which the polynucleotide is operatively associated with a promoter, wherein the vectors are capable of expressing an antibody or antigen-binding fragment thereof that specifically binds Siglec-15 in a suitable host cell.

Also disclosed is a method of producing an antibody or fragment thereof that specifically binds Siglec-15, comprising culturing a host cell containing a vector comprising the polynucleotides described above and recovering said antibody or fragment thereof. Isolated antibodies or fragments thereof produced by the method described above are disclosed herein.

As used herein, "host cells" refers to cells that harbor vectors constructed using recombinant DNA techniques and that encode at least one heterologous gene. In descriptions of processes for the isolation of antibodies from recombinant hosts, the term "cell" and the term "cell culture" are used interchangeably to indicate the source of antibodies, unless otherwise clearly specified. In other words, recovery of the polypeptide from "cells" can mean both spun whole cells and cell culture containing both medium and suspended cells.

A variety of host expression vector systems can be used to express antibody molecules for use in the methods described herein. Such host expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells that, when transformed or transfected with the coding sequences of suitable nucleotides, can express an antibody molecule of the disclosure in situ. These include, but are not limited to, microorganisms such as bacteria (eg, E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (eg, Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (eg, baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg, Ti plasmid) that contain antibody coding sequences; and mammalian cell systems (eg, COS, CHO, BLK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (eg, metallothionein promoter) or from mammalian viruses (eg, the late adenovirus promoter; the 7.5K vaccinia virus promoter). For the expression of a recombinant antibody molecule, bacterial cells, such as Escherichia coli, or eukaryotic cells are used, especially for the expression of a complete recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, together with a vector such as the human cytomegalovirus major intermediate early gene promoter element is an efficient expression system for antibodies (Foecking et al. , Gene 45:101 (1986);Cockett et al., Bio/Technology 8:2 (1990)).

The host cell line used for protein expression is often of mammalian origin; those skilled in the art are well able to determine the particular host cell lines that are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, CHO (Chinese hamster ovary), DG44 and DUXB11 (Chinese hamster ovary lines, DHFr minus), HELA (human cervical carcinoma), CVI (monkey kidney line) , COS (a derivative of CVI with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblast), BALBC/3T3 (mouse fibroblast), HAK (line of hamster kidney), SP2/O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney). Host cell lines are usually available from commercial services, the American Tissue Culture Collection, or the published literature.

In addition, a host cell strain can be chosen that modulates the expression of the inserted sequences, or that modifies and processes the gene product in the specific manner desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important to protein function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure correct modification and processing of the foreign protein expressed. For this purpose, eukaryotic host cells can be used that possess the cellular machinery for the appropriate processing of primary transcription, glycosylation, and phosphorylation of the gene product.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the antibody molecule can be engineered. Instead of using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by suitable expression control elements (eg, promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc. ) and a selectable marker. After introduction of the foreign DNA, the genetically engineered cells can be allowed to grow for 1-2 days in an enriched medium and then switched to a selective medium. The selectable marker on the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that can in turn be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that stably express the antibody molecule.

A number of selection systems can be used including, but not limited to, the herpes simplex virus thymidine kinase genes (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc Natl. Acad. Sci. USA 48:202 (1992)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 1980) which can be used in tk-, hgprt- or aprt- cells, respectively. In addition, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al. al., Proc. Natl. Acad. Sci. USA 78:1527 (1981 )); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev Pharmacol. Toxicol.

32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); TIB TECH 11 (5): 155-215 (May, 1993); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984). Methods commonly known in the art of recombinant DNA technology that can be used are described in Ausubel et al. (eds.) , Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993), Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990), and in Chapters 12 and 13, Dracopoli et al (eds. ), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994), Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981).

Expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987)). When a marker in the antibody expressing vector system is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region associates with the antibody gene, antibody production will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

In vitro production allows for scale-up to provide large quantities of the desired polypeptides. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include culture in homogeneous suspension, for example, in an air reactor or continuous stirred reactor, or culture of immobilized or entrapped cells. eg in hollow fibers, microcapsules, in agarose microbeads or in ceramic cartridges. If necessary and/or desired, the polypeptide solutions can be purified by standard chromatography methods, for example gel filtration, ion exchange chromatography, DEAE-cellulose chromatography or (immuno)affinity chromatography. , for example, after preferential biosynthesis of a synthetic hinge region polypeptide or before or after the HIC chromatography step described herein.

Constructs encoding anti-Siglec-15 antibodies or fragments, variants or derivatives thereof, as disclosed herein, can also be expressed in non-mammalian cells, such as bacteria or yeast or plant cells. Bacteria that readily take up nucleic acids include members of the Enterobacteriaceae, such as Escherichia coli or Salmonella strains; bacillacea, such as Bacillus subtilis; pneumococcus; Streptococcus and Haemophilus influenzae. Furthermore, it is appreciated that, when expressed in bacteria, heterologous polypeptides typically form part of inclusion bodies. Heterologous polypeptides must be isolated, purified, and then assembled into functional molecules. When tetravalent forms of antibodies are desired, the subunits will self-assemble into tetravalent antibodies (WO02/096948A2).

In bacterial systems, a number of expression vectors may be advantageously selected depending on the intended use of the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors that direct the expression of high levels of fusion protein products that are purified may be desirable. easily. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence can be individually ligated into the vector at phase with the lacZ coding region to produce a fusion protein; pIN vectors (Inouye and Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke and Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to a matrix of glutathione-agarose beads, followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In addition to prokaryotes, eukaryotic microbes can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms, although there are a number of other strains commonly available, such as Pichia pastoris.

For expression in Saccharomyces, the plasmid YRp7 is commonly used, for example (Stinchcomb et al., Nature 282:39 (1979); Kingsman et al., Gene 7:141 (1979); Tschemper et al., Gene 10 :157 (1980)). This plasmid already contains the TRP1 gene which provides a selection marker for a mutant strain of yeast that lacks the ability to grow in tryptophan, eg, ATCC #44076 or PEP4-1 (Jones, Genetics 85:12 ( 1977)). The presence of the trpl lesion as a feature of the yeast host cell genome then provides an efficient environment for detecting transformation by growth in the absence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is commonly used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence can be individually cloned into non-essential regions (eg, the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (eg, the polyhedrin promoter).

Once the anti-Siglect-15 antibody or fragment, variant or derivative thereof, as disclosed herein, has been recombinantly expressed, it can be purified by any method known in the art for the purification of a Siglec-15 molecule. immunoglobulin, for example, by chromatography (eg, ion exchange, affinity, particularly by affinity for the specific antigen after protein A and sizing column chromatography), centrifugation, differential solubility, or by any other conventional technique for purification of proteins.

SAW. PHARMACEUTICAL COMPOSITIONS COMPRISING ANTI-SIGLEC-15 ANTIBODIES OR ANTIGEN-BINDING FRAGMENTS THEREOF

The pharmaceutical compositions used in the present disclosure comprise pharmaceutically acceptable carriers well known to those skilled in the art. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Certain pharmaceutical compositions disclosed herein may be administered orally in an acceptable dosage form including, for example, capsules, tablets, aqueous suspensions, or solutions. Certain pharmaceutical compositions can also be administered by nasal spray or inhalation. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like may also be present. Formulations suitable for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., 16th ed. (1980).

The amount of an anti-Siglec-15 antibody or immunoconjugate, which can be combined with the carrier materials to produce a single dosage form, will vary depending on the host treated and the particular mode of administration. Dosage schedules can also be adjusted to provide the optimal desired response. The compositions may also comprise the Siglec-15 immunoconjugates, antibodies or fragments, variants or derivatives thereof dispersed in a biocompatible carrier material that acts as a suitable carrier or delivery system for the compounds.

VII. TREATMENT METHODS USING IMMUNOCONJUGATES, ANTIBODIES OR ANTIGEN-BINDING FRAGMENTS OF THE SAME THERAPEUTICS

Methods of preparing and administering an immunoconjugate, antibody or fragment, variant or derivative thereof against Siglec-15, as disclosed herein, to a subject in need thereof are well known or readily determined by those skilled in the art. matter.

The Siglec-15 immunoconjugates, antibodies, or antigen-binding fragments thereof of the disclosure are useful for the treatment of a leukemia, such as AML. In certain disclosures, immunoconjugates, antibodies, or antigen-binding fragments thereof against Siglec-15 advantageously reduce side effects associated with conventional leukemic therapies.

Leukemias are cancers that originate in the bone marrow, where the malignant cells are white blood cells (leukocytes). Acute myelogenous leukemia (also called acute myelocytic leukemia, acute myeloblastic leukemia, acute granulocytic leukemia, and acute nonlymphocytic leukemia) is a malignancy that arises in granulocytes or monocytes. AML is characterized by the uncontrolled overgrowth and accumulation of cells called leukemic blasts, which fail to act like normal blood cells and block the production of normal marrow cells, leading to a deficiency of red blood cells ( anemia) and platelets (thrombocytopenia) and normal white blood cells (especially neutrophils, i.e. neutropenia) in the blood.

CD33-targeted therapies, such as GO, produce significant side effects, including infusion syndrome, when administered to patients with AML. Therefore, the ability to target Siglec-15 for therapy provides an alternative to CD33-based treatments. On the other hand, since the leukemic blasts are anti-Siglec-15 both CD33+ and CD33- antibodies or antigen-binding fragments thereof provide an additional treatment option for AML. This is important for several reasons. Improving cell killing can increase the percentage of complete remissions, increase the time to remission, and decrease the likelihood of relapse. Second, improved cell killing may decrease the overall need for toxin therapy, resulting in decreased nonspecific cell killing and side effects. Third, for those patients with AML who have relatively low expression of CD33, either naturally or due to growth of CD33- cells after undergoing CD33 therapy, antibodies against Siglec-15 or binding fragments to antigen thereof are a valuable alternative or combination therapy.

On the other hand, since the surface expression of Siglec-15 is low in peripheral blood leukocytes from normal control donors and high in blast cells from AML patients, side effects of administration of immunoconjugates, antibodies or fragments Their antigen-binding activity against Siglec-15 will be reduced compared to conventional leukemia therapies.

All subtypes of AML are suitable for treatment with an antibody against Siglec-15 or an antigen-binding fragment thereof. AML subtypes are classified based on the stage of development that the myeloblasts have reached at the time of diagnosis. The categories and subsets allow the doctor to decide which treatment works best for the cell type and how quickly the disease may develop. The subsets are: M0, myeloblastic, under special analysis; M1, Myeloblastic, without maturation; M2, Myeloblastic, with maturation; M3, Promyelocytic; M4, Myelomonocytic; M5, Monocytic; M6, Erythroleukemia; and M7, Megakaryocytic. An antibody against Siglec-15 or antigen-binding fragment thereof can be administered with a secondary agent that is particularly suitable for the AML subtype. For example, acute promyelocytic leukemia (APL) and acute monocytic leukemia are subtypes of AML that require different treatment than other subtypes of AML. A second agent for the treatment of APL may include all-trans retinoic acid (ATRA) or an antimetabolite, such as like cytarabine. A second agent for the treatment of acute monocytic leukemia may include a deoxyadenosine analog, such as 2-chloro-2'-deoxyadenosine (2-CDA).

Risk factors for AML include the presence of certain genetic disorders, such as Down syndrome, Fanconi anemia, Shwachman-Diamond syndrome, and others. An AML patient with a genetic disorder may be administered an antibody to Siglec-15 or antigen-binding fragment thereof and a second agent to treat a symptom of the genetic disorder. For example, a patient with AML and Fanconi anemia may be given an antibody against Siglec-15 or antigen-binding fragment thereof and an antibiotic.

Other risk factors for AML include chemotherapy or radiation therapy for a different cancer, tobacco smoke, and exposure to large amounts of benzene.

Immunoconjugates, antibodies, or antigen-binding fragments thereof against Siglec-15 can be administered to a human or other animal according to the aforementioned methods of treatment in an amount sufficient to produce a therapeutic effect. With respect to AML, therapy can be considered effective if there is a statistically significant difference in the rate or proportion of malignant cells in the bloodstream or bone marrow. Therapy is considered to be effective, for example, when remission is achieved, which is when there are no signs of malignant cells.

The efficacy of administering a first agent and, optionally, a second agent can also be evaluated based on, for example, a decrease in the number of malignant cells found in the bloodstream, a decrease in the frequency or severity of infection bacterial or viral infection, increased speed of wound healing, and the general feeling of the patient, including increased energy level and decreased pain in the bones and joints.

The route of administration of the anti-Siglec-15 immunoconjugate, antibody or fragment, variant or derivative thereof, may be, for example, oral, parenteral, inhalation or topical. The term parenteral as used herein includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, or subcutaneous administration. A suitable form of administration would be a solution for injection, in particular for intravenous or intra-arterial injection or drip.

Anti-Siglec-15 immunoconjugates, antibodies, or antigen-binding fragments thereof can be administered on multiple occasions and with various frequencies, depending on various factors known to those of skill in the art. Alternatively, the anti-Siglec-15 immunoconjugates, antibodies, or antigen-binding fragments thereof may be administered in the form of a sustained-release formulation, in which case less frequent administration is required. The dose and frequency vary depending on the half-life of the immunoconjugate, antibody, or antigen-binding fragment in the patient.

The practice of the disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are widely explained in the literature. See, eg, Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold Spring Harbor Laboratory Press: (1989); Molecular Cloning: A Laboratory Manual, Sambrook et al., ed., Cold Springs Harbor Laboratory, New York (1992), DNA Cloning, D. N. Glover ed., Volumes I and II (1985); Oligonucleotide Synthesis, M. J. Gait ed., (1984); Mullis et al. Pat. US No.: 4,683,195; Nucleic Acid Hybridization, B.D. Hames & S.J. Higgins eds. (1984); Transcription And Translation, B.D. Hames & S.J. Higgins eds. (1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., (1987); Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); treatise, Methods In Enzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors For Mammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory (1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds); Immunochemical Methods In Cell And Molecular Biology, Mayer and Walker, eds., Academic Press, London (1987); Handbook Of Experimental Immunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., (1986); Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989).

The general principles of antibody engineering are set forth in Antibody Engineering, 2nd edition, C.A.K. Borrebaeck, Ed., Oxford Univ. Press (1995). General principles of protein engineering are set forth in Protein Engineering, A Practical Approach, Rickwood, D., et al., Eds., IRL Press in Oxford Univ. Press, Oxford, Ing. (1995). General principles of antibodies and antibody-hapten binding are set forth in: Nisonoff, A., Molecular Immunology, 2nd ed., Sinauer Associates, Sunderland, MA (1984); and Steward, M.W., Antibodies, Their Structure and Function, Chapman and Hall, New York, NY (1984). Furthermore, standard methods in immunology known in the art and not specifically described are generally followed as in Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al. (eds), Basic and Clinical -Immunology (8th ed.), Appleton & Lange, Norwalk, CT (1994) and Mishell and Shiigi (eds), Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York (1980).

Conventional reference works that set forth the general principles of immunology include Current Protocols in Immunology, John Wiley & Sons, New York; Klein, J., Immunology: The Science of Self-Nonself. They Discriminated, John Wiley & Sons, New York (1982); Kennett, R., et al., eds., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, New York (1980); Campbell, A., "Monoclonal Antibody Technology" in Burden, R., et al., eds., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Elsevere, Amsterdam (1984), Kuby Immunnology 4th ed. Ed. Richard A. Goldsby, Thomas J. Kindt, and Barbara A. Osborne, H. Freemand & Co. (2000); Roitt, I., Brostoff, J., and Male D., Immunology 6th ed. London: Mosby (2001); Abbas A., Abul, A., and Lichtman, A., Cellular and Molecular Immunology Ed. 5, Elsevier Health Sciences Division (2005); Kontermann and Dubel, Antibody Engineering, Springer Verlan (2001); Sambrook and Russell, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press (2001); Lewin, Genes VIII, Prentice Hall (2003); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988); Dieffenbach and Dveksler, PCR Primer Cold Spring Harbor Press (2003).

EXAMPLES

Example 1: Multiple Splicing and Induction of Siglec-15 Transcription in Leukemic Cell Lines

Four splice variants of human Siglec-15 were identified by RT-PCR using specific primers. Electrophoresis of RT-PCR products from DAMI (megakaryocytic cell line), HEL (erythroleukemic cell line), and K562 (erythroleukemic cell line) are shown in Figure 1A with exon maps generated after sequencing (Figure 1A - panel law). Four different splice variants of Siglec-15 were identified from RT-PCR of cDNA from leukemic cell lines. The largest transcript (2D) comprised all exons and was used in other experiments.

The Siglec-15 transcript was also found in 6 of the 7 different cell lines tested (FIG. 1B). Only YT (natural killer cell-like leukemic cell line) did not show any Siglec-15 transcripts. Stimulation with phorbomeristyl acetate (PMA) or lipopolysaccharide (LPS) for 48 hours upregulated transcript expression. GAPDH RT-PCR is shown below each panel to control for equal charge (0.4 kb). 293T cells were used as a negative control.

RT-PCR was also performed to identify the full-length Siglec-15 transcript in 7 cell lines derived from leukemic patients (Figure 1B). These cell lines were also stimulated with LPS or PMA for 48 hours. Expression of the Siglec-15 transcript was found in 6 of the 7 cell lines and upregulation was observed after stimulation in all cases except the CHRF-288-11 cell line. The full-length transcript was the only one consistently upregulated. In the HL60 cell line, while PMA induced upregulation of the full-length transcript, LPS stimulation resulted in complete loss of this transcript. These results demonstrate that Siglec-15 exists in multiple splice variants and that the full-length transcript is inducible by LPS and PMA in many leukemic cell lines.

Example 2: Antibodies against Siglec-15 or antigen-binding fragments thereof

Phage display was used to select for reagents that specifically bind Siglec-15. Single-chain variable fragment (ScFv) binders to purified Flag-His-tagged Siglec-15 were amplified using phage display technology and subsequently selected on the surface of CHO cells stably expressing HA-tagged Siglec-15 cloned on a pDisplay construction. A9E8 and A4C9 were two ScFv binders from these two rounds that were converted to the murine IgG1 format. Both bound Siglec-15 by FACS (Figure 2A) and confocal microscopy (Figure 2B) and A4C9, also bound on Western blots (Figure 2D).

These two antibodies are unlikely to cross-react with other Siglecs, as the similarity between Siglec-15 and other Siglecs is very low (less than 35% identity). To confirm the specificity of A9E8 and A4C9 for Siglec-15, the ScFv versions of the two antibodies were compared for their binding to CHO cells stably transfected with HA-labeled Siglec-15, Siglec-11, Siglec-16 or non-transfected CHO cells. These two antibodies bound strongly to Siglec-15, while no binding was observed for Siglec-11 and 16 transfectants (Figure 2C). U937 is a well-characterized cell line that endogenously expresses many Siglecs on its cell surface, including CD33, Siglec-5, and Siglec-9 (Nguyen DH et al 2006). A9E8 did not bind to the surface of U937 cells (FIG. 5A), again consistent with the lack of cross-reactivity with other Siglecs.

Example 3: Expression patterns of Siglec-15

Endogenous Siglec-15 has previously been shown to be predominantly intracellular by immunohistochemistry (Angata et al 2007). This finding was confirmed by FACS analysis of unfixed as well as fixed and permeabilized leukemic cell lines, although these lines differed in the relative levels of surface and intracellular expression (FIG. 3). The low surface expression can be explained by the need for a membrane adapter protein, such as DAP12 and DAP10 (Angata et al 2007). To test the need for a membrane adapter protein, Flag-tagged Siglec-15 was transiently transfected into HEK-293 cell lines stably transfected with different adapter molecules: DAP12, DAP10 or FcRy. HEK-293 cells were chosen because they lack endogenous expression of these adapters. Surface expression of Flag-Siglec-15 was detected using an anti-Flag monoclonal antibody by FACS. Siglec-15 surface enhancement was more notable in the presence of DAP10, but significant expression was also observed in the presence of DAP12 and FcRy (Figure 4). A parallel experiment using Siglec-16 showed a more specific DAP12-dependent surface expression (FIG. 4). These results demonstrate that Siglec-15 is a prominent intracellular antigen under normal growth conditions, but some can access the cell surface, in conjunction with an adaptor. The apparent promiscuity of Siglec-15's dependence on adapters for surface expression differentiates it from other activating receptors, which bind to an adapter more specifically.

The A9E8 antibody was used to examine the surface expression of Siglec-15 in leukemic cell lines. By FACS, Siglec-15 was identified on the cell surface of leukemic cell lines of the erythroid (K562 and HEL) and megakaryocytic (DAMI) lineages (Figure 5A). Monocytic (U937 and SKM-1) and promonocytic (NOMO-1) leukemic cell lines did not show any cell surface expression (Figure 5A). Leukemic cell lines of lymphoid lineage were also tested. Only Daudi (B-cell lymphoma) showed relatively weak surface expression (FIG. 5A). There was no expression in Jurkat T cell lymphoblastic leukemia (FIG. 5A).

Western blot analysis of selected cell lines using monoclonal antibody A4C9 showed an expression pattern similar to that of a rabbit antiserum, 1A, specific for the cytoplasmic tail of Siglec-15, which bound to Flag-tagged Siglec-15. transfectant into 293T cells (Figure 5B and 5C). Western blots revealed significant expression of Siglec-15 protein in Jurkat cells (band indicated by single arrows) and weaker expression in K562 and HEL cell lines (Figure 5C).

Example 4: Surface expression of Siglec-15 in peripheral blood leukocytes from healthy donors

Peripheral blood cells from healthy donors were used to study the expression of Siglec-15. Siglec-15 was absent from the surface of most mature leukocytes tested: T cells (CD3+), B cells (CD19+), and NK cells (CD56+), but rare populations (<1%) of monocytes (CD14+) showed surface expression of Siglec-15 (FIG. 6A). This pattern of expression was similar in six healthy donors tested.

Siglec-15 was previously reported to be intracellular in macrophages and dendritic cells by immunohistochemistry. FACS analysis showed very low levels of surface expression of Siglec-15 in 7% of cultured macrophages derived from peripheral blood monocytes (FIG. 6B). Siglec-15 staining was more prominent in forward low scatter cells, probably corresponding to a less mature subpopulation of macrophages. Surface staining in macrophages was lost after 24 hours of LPS treatment (FIG. 6B). Dendritic cells showed extremely low Siglec-15 surface expression in ~4% of the total population before LPS stimulation (FIG. 6B). This expression was also lost after 24 hours of LPS stimulation (FIG. 6B). These results demonstrate that, in healthy donors, Siglec-15 is scarce or absent on the cell surface of most circulating leukocytes in the blood, as well as on macrophages and monocyte-derived dendritic cells.

Example 5: Surface expression of Siglec-15 in circulating AML blasts

In contrast to healthy donors, significantly higher surface expression of Siglec-15 was found in blasts from 8 of 10 AML patients tested (Figure 7A-D). The average size of Siglec-15 positive blast subpopulations was 20%, compared to -2% for circulating peripheral blood leukocytes from healthy donors (Figure 7E). Figure 7 summarizes four representative examples of the group of AML donors studied. Patients A (FIG. 7A), B (FIG. 7B), and C (FIG. 7C) consistently demonstrated co-expression of Siglec-15 with CD14.

CD33 is expressed on 90% of AML myeloblasts. In one case, patient C (FIG. 7C), who was CD33-negative, a high level of Siglec-15 was expressed in 20% of the blasts. In another case, patient D showed a low percentage of CD33+ blasts (7%) but a higher proportion of Siglec-15+ blasts (15%) (data not shown). HLA-DR was not consistently coexpressed with Siglec-15, except in a few patients, such as A. Patient D expressed more Siglec-15 in the larger blasts, as defined by greater forward scatter. These data indicate that Siglec-15 is significantly more expressed in circulating white blood cells from AML patients than in those from healthy donors.

Example 6: A9E8 Induces an Extremely Fast Endocytosis Rate of Surface Siglec-15

A rapid rate of endocytosis of Siglec-15 would be advantageous, especially if a toxin-conjugated antibody were used to target Siglec-15-positive AML blasts. Rapid endocytosis of Siglec-15 is expected from its cytoplasmic tail sequence, which encodes an ITSM motif of SNYENL sequence and thus fits the classic YxxO endocytosis motif, where X is any amino acid and O is a hydrophobic moiety. The YxxO motif directs clathrin-dependent endocytosis of membrane proteins (Collawn et al 1990) by binding to the AP2 clathrin adapter complex (Aguilar et al 2001; Boehm and Bonifacino, 2001). To investigate the rate of endogenous endocytosis of Siglec-15 in vitro, the K562 cell line was chosen because it shows significant surface expression. The A9E8 antibody bound to K562 on ice and the cells were resuspended in medium prewarmed at 37°C. The cells The heated cells were immediately incubated for various times at 37°C, before cooling with 5 ml ice-cold FACS buffer. A secondary antibody was used to detect the remaining surface antibody and the endocytosis half-life was measured as 174 seconds. To monitor the natural dissociation of the A9E8 antibody from Siglec-15, a pDisplay construct encoding only the extracellular domains of Siglec-15 was subjected to the same assay. Lacking the natural transmembrane and cytoplasmic tail of Siglec-15, this construct showed no apparent surface endocytosis during the first fifteen minutes of incubation at 37°C. This result demonstrates that the disappearance of surface A9E8 staining on K562 cells is probably due to endocytosis.

Claims (13)

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to Siglec-15 comprising the sequences of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of Table 1 corresponding to SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively. 2. The isolated antibody or antigen-binding fragment thereof of claim 1 comprising (a) a VH sequence at least 90%, 95% or 100% identical to SEQ ID NO: 1 of Table 1; and (b) a VL sequence at least 90%, 95%, or 100% identical to SEQ ID NO: 2 of Table 1. 3. The antibody or antigen-binding fragment thereof of any of claims 1-2, which is humanized, chimeric, fully human, a Fab fragment, a Fab' fragment, an F(ab)2 fragment or an Fv fragment single chain (scFv). 4. An isolated cell capable of producing the antibody or antigen-binding fragment thereof of any one of claims 1-3. 5. A method of manufacturing the antibody or antigen-binding fragment thereof of any one of claims 1-3, comprising (a) culturing the cell of claim 4 and (b) isolating said antibody or antigen-binding fragment thereof antigen thereof from said cultured cell. 6. An immunoconjugate having the formula (A) -(L) -(C) or (C) -(L) -(A), wherein: (A) is an antibody or antigen-binding fragment thereof of any one of claims 1-3; (L) is a linker; Y (C) is a cytotoxic agent; Y wherein said linker (L) joins (A) to (C). 7. A composition comprising the antibody or antigen-binding fragment thereof, or immunoconjugate of any one of claims 1-3 or 6 and a carrier. 8. An isolated polynucleotide comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-3. 9. A vector comprising the polynucleotide of claim 8. 10. An isolated antibody or antigen-binding fragment thereof that specifically binds Siglec-15 according to any one of claims 1 to 3 or an immunoconjugate according to claim 6 for use in the treatment of leukemia acute myeloid. 11. An isolated antibody or antigen-binding fragment thereof that specifically binds Siglec-15 according to any one of claims 1 to 3 or an immunoconjugate according to claim 6 for use in the treatment of leukemia acute myeloid cancer (AML), where the treatment reduces the side effects of AML treatment associated with conventional leukemic therapies. 12. An isolated antibody or antigen-binding fragment thereof that specifically binds Siglec-15 according to any one of claims 1 to 3 or an immunoconjugate according to claim 6 for use in the treatment of AML reducing the number of blasts in a patient with AML. 13. An isolated antibody or antigen-binding fragment thereof that specifically binds Siglec-15 according to any one of claims 1 to 3 or an immunoconjugate according to claim 6 for use in the treatment of leukemia acute myeloid in a subject who has low CD33 expression or who is CD33 negative.